Julie A. Johnson

The relationship between fractures and DXA measures of BMD in the distal femur of children and adolescents with cerebral palsy or muscular dystrophy

Children with limited or no ability to ambulate frequently sustain fragility fractures. Joint contractures, scoliosis, hip dysplasia, and metallic implants often prevent reliable measures of bone mineral density (BMD) in the proximal femur and lumbar spine, where BMD is commonly measured. Further, the relevance of lumbar spine BMD to fracture risk in this population is questionable. In an effort to obtain bone density measures that are both technically feasible and clinically relevant, a technique was developed involving dual‐energy X‐ray absorptiometry (DXA) measures of the distal femur projected in the lateral plane. The purpose of this study is to test the hypothesis that these new measures of BMD correlate with fractures in children with limited or no ability to ambulate. The relationship between distal femur BMD Z‐scores and fracture history was assessed in a cross‐sectional study of 619 children aged 6 to 18 years with muscular dystrophy or moderate to severe cerebral palsy compiled from eight centers. There was a strong correlation between fracture history and BMD Z‐scores in the distal femur; 35% to 42% of those with BMD Z‐scores less than −5 had fractured compared with 13% to 15% of those with BMD Z‐scores greater than −1. Risk ratios were 1.06 to 1.15 (95% confidence interval 1.04–1.22), meaning a 6% to 15% increased risk of fracture with each 1.0 decrease in BMD Z‐score. In clinical practice, DXA measure of BMD in the distal femur is the technique of choice for the assessment of children with impaired mobility.

Immuno-localization of the calcitriol receptor, calbinclin-D28k and the plasma membrane calcium pump in the human eye

Using immunohistochemical methods, we detected epitopes of the calcitriol receptor, the ATP-dependent plasma membrane calcium pump and the 28kD vitamin D-dependent calcium-binding protein in sections of the human eye. In retinal photoreceptors, vitamin D receptor, plasma membrane calcium pump and calcium-binding protein epitopes were detected in the outer nuclear layer. Epitopes for the vitamin D receptor and the calcium-binding protein were present in the inner and outer segments of the photoreceptors, where visual transduction occurs. All three proteins were detected in some cells of the ganglion cell layer, the inner nuclear layer, and the retinal pigment epithelium. Epitopes for these proteins also were noted in the ciliary body epithelium. VDR epitopes were seen in lens epithelium. Some immunostaining for VDR, PMCA and calbindin-D28k also was present in the endothelium and in the basal epithelium of the cornea. The presence of these proteins in several tissues of the human eye suggests that the proteins may play a role in the cellular physiology of the eye. Their exact functions in the eye remain undetermined.

1,25-Dihydroxyvitamin D3 receptors in developing dorsal root ganglia of fetal rats

We observed immunostaining for the 1,25-dihydroxyvitamin D(3) receptor (VDR) and calbindin-D(28k) in neurons, but not glial cells, of fetal rat dorsal root ganglia (DRG) from days 13 through 21 of gestation. Dispersed cultures of DRG collected from rat fetuses at gestational day 15 also contained epitopes for VDR and calbindin-D(28k) in neurons, but not in glial cells. 1,25-Dihydroxyvitamin D(3), through VDR, may perform significant functions in the development of neuronal cells.

Very important pharmacogene summary: thiopurine S-methyltransferase

Thiopurine S-methyltransferase (TPMT, S-adenosyl-L-methionine : thiopurine S-methyltransferase; EC 2.1.1.67) catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine (6-MP) and azathioprine as well as other aromatic and heterocyclic sulfhydryl compounds [1,2].Weinshilboum and Sladek

Vitamin D and renal calcium transport.

The kidney plays an important role in maintaining calcium balance in plasma and extracellular fluid. 1,25 Dihydroxyvitamin D3, which regulates intestinal calcium absorption, bone calcium resorption, and renal calcium reabsorption, is synthesized in the renal proximal tubules. Vitamin D-dependent calcium reabsorption from urine occurs in renal distal tubules. New information, obtained from both intestinal and kidney cells, enables us to propose a model of the mechanism of vitamin D-dependent calcium transport in renal distal tubule cells. This model involves several vitamin D-regulated processes and proteins. Briefly, 1,25 dihydroxyvitamin D3 stimulates calcium uptake at the distal tubule luminal membrane. A cytosolic vitamin D-dependent calcium binding protein (calbindin-D) sequesters calcium, which allows more calcium to enter the cell and more efficient diffusion of calcium across the cell. The ATP-dependent plasma membrane Ca2+ pump in the basolateral membrane pumps calcium out of the distal tubule cells into plasma.

Very important pharmacogene summary: sulfotransferase 1A1

Department of Genetics, Stanford University, Stanford, California, USA and Division of Clinical Pharmacology, Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic-Mayo Medical School, Rochester, Minnesota, USA Correspondence to Teri E. Klein, Department of Genetics, Stanford University, 300 Pasteur Drive, L-301, Stanford, CA 94305, USA Tel: + 1 650 736 0156; e-mail: teri.klein@stanford.edu

Quantifying weight-bearing by children with cerebral palsy while in passive standers

Purpose: Children who are nonambulatory are placed into standers with the goal of providing benefits from weight-bearing. The purpose of this study was to quantify weight-bearing loads by children with cerebral palsy while in standers. Methods: Electronic load-measuring footplates were fabricated specifically for this study. Weight-bearing loads were continuously measured in 19 children who were nonambulatory during routine 30-minute standing sessions (3–6 sessions/child, total 110 sessions). Results: Weight-bearing ranged widely (23%–102%) with a mean of 68% of body weight. There was some variation over the course of a session and between different sessions, but more variance was noted between subjects. Conclusions: Actual weight borne in a stander is quite variable, and in some instances only a fraction of actual body weight. Further studies are required to delineate relevant factors and identify ways to maximize weight-bearing loads while in a stander.

A novel application of pattern recognition for accurate SNP and indel discovery from high-throughput data: Targeted resequencing of the glucocorticoid receptor co-chaperone FKBP5 in a Caucasian population

The detection of single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) with precision from high-throughput data remains a significant bioinformatics challenge. Accurate detection is necessary before next-generation sequencing can routinely be used in the clinic. In research, scientific advances are inhibited by gaps in data, exemplified by the underrepresented discovery of rare variants, variants in non-coding regions and indels. The continued presence of false positives and false negatives prevents full automation and requires additional manual verification steps. Our methodology presents applications of both pattern recognition and sensitivity analysis to eliminate false positives and aid in the detection of SNP/indel loci and genotypes from high-throughput data. We chose FK506-binding protein 51(FKBP5) (6p21.31) for our clinical target because of its role in modulating pharmacological responses to physiological and synthetic glucocorticoids and because of the complexity of the genomic region. We detected genetic variation across a 160 kb region encompassing FKBP5. 613 SNPs and 57 indels, including a 3.3 kb deletion were discovered. We validated our method using three independent data sets and, with Sanger sequencing and Affymetrix and Illumina microarrays, achieved 99% concordance. Furthermore we were able to detect 267 novel rare variants and assess linkage disequilibrium. Our results showed both a sensitivity and specificity of 98%, indicating near perfect classification between true and false variants. The process is scalable and amenable to automation, with the downstream filters taking only 1.5h to analyze 96 individuals simultaneously. We provide examples of how our level of precision uncovered the interactions of multiple loci, their predicted influences on mRNA stability, perturbations of the hsp90 binding site, and individual variation in FKBP5 expression. Finally we show how our discovery of rare variants may change current conceptions of evolution at this locus.

Immunolocalization of Calcitriol Receptor, Plasma Membrane Calcium Pump and Calbindin-D28k in the Cornea and Ciliary Body of the Rat Eye

Epitopes of the calcitriol receptor, the ATP-dependent plasma membrane calcium pump (PMCA) and the 28-kD vitamin-D-dependent Ca-binding protein (calbindin-D28k) were detected in sections of the albino rat eye using light microscopy and immunohistochemistry of paraffin-embedded tissues. Calcitriol receptor and calbindin-D28k epitopes were detected in both the inner and outer layers of the ciliary body epithelium. PMCA was present in both epithelial cell layers of the ciliary body but was most prominent in the inner layer. Corneal epithelium and endothelium also contained epitopes for calcitriol receptor, PMCA and calbindin-D28k. These Ca-regulatory proteins may play a role in the cellular physiology of the albino rat eye by maintaining appropriate intracellular and aqueous humor Ca concentrations.

Comparison of Whole-Genome Sequencing Methods for Analysis of Three Methicillin-Resistant Staphylococcus aureus Outbreaks

ABSTRACT Whole-genome sequencing (WGS) can provide excellent resolution in global and local epidemiological investigations of Staphylococcus aureus outbreaks. A variety of sequencing approaches and analytical tools have been used; it is not clear which is ideal. We compared two WGS strategies and two analytical approaches to the standard method of SmaI restriction digestion pulsed-field gel electrophoresis (PFGE) for typing S. aureus. Forty-two S. aureus isolates from three outbreaks and 12 reference isolates were studied. Near-complete genomes, assembled de novo with paired-end and long-mate-pair (8 kb) libraries were first assembled and analyzed utilizing an in-house assembly and analytical informatics pipeline. In addition, paired-end data were assembled and analyzed using a commercial software package. Single nucleotide variant (SNP) analysis was performed using the in-house pipeline. Two assembly strategies were used to generate core genome multilocus sequence typing (cgMLST) data. First, the near-complete genome data generated with the in-house pipeline were imported into the commercial software and used to perform cgMLST analysis. Second, the commercial software was used to assemble paired-end data, and resolved assemblies were used to perform cgMLST. Similar isolate clustering was observed using SNP calling and cgMLST, regardless of data assembly strategy. All methods provided more discrimination between outbreaks than did PFGE. Overall, all of the evaluated WGS strategies yielded statistically similar results for S. aureus typing.