Multi-laboratory validation of a Δ9-tetrahydrocannabinol LC-MS/MS test kit designed for quantifying THC and marijuana metabolites in blood.

Marijuana legalization has increased the demand for testing of Δ9-tetrahydrocannabinol (THC) and THC metabolites. The THC ToxBox® test kit (THC ToxBox®) is commercially available and supports high-throughput LC-MS/MS analytical methods designed to quantify low levels of THC and THC metabolites in blood. The purpose of this study is to determine if this new test kit meets the rigors of laboratory accreditation and produces equivalent results across six states- and locally-funded laboratories. Each laboratory followed internal method validation procedures established for their clinical (CLIA) or international (ISO17025) accreditation program. Test performance indicators included accuracy, precision, measurement of uncertainty, calibration models, reportable range, sensitivity, specificity, carryover, interference, ion suppression/enhancement and analyte stability. Analytes and interferents were resolved within the 6-min analytical runtime, and the 48-well plate pre-manufactured with calibrators, second source quality control material, and internal standards at precise concentrations allowed for simple and consistent sample preparation in less than one hour. Every laboratory successfully validated test kit procedures for forensic use. Differences in sensitivity were generally associated with the use of older equipment. Statistical analysis of results spanning reportable ranges show that laboratories with different instrument platforms produce equivalent results at levels sufficiently low enough to support per se limit testing of THC and THC metabolites (1-5 ng/mL). THC ToxBox® represents a viable option for state- and locally-funded laboratories charged with investigating impaired driving cases involving marijuana use.

A PAX1 enhancer locus is associated with susceptibility to idiopathic scoliosis in females.

Idiopathic scoliosis (IS) is a common paediatric musculoskeletal disease that displays a strong female bias. By performing a genome-wide association study (GWAS) of 3,102 individuals, we identify significant associations with 20p11.22 SNPs for females (P=6.89 × 10(-9)) but not males (P=0.71). This association with IS is also found in independent female cohorts from the United States of America and Japan (overall P=2.15 × 10(-10), OR=1.30 (rs6137473)). Unexpectedly, the 20p11.22 IS risk alleles were previously associated with protection from early-onset alopecia, another sexually dimorphic condition. The 174-kb associated locus is distal to PAX1, which encodes paired box 1, a transcription factor involved in spine development. We identify a sequence in the associated locus with enhancer activity in zebrafish somitic muscle and spinal cord, an activity that is abolished by IS-associated SNPs. We thus identify a sexually dimorphic IS susceptibility locus, and propose the first functionally defined candidate mutations in an enhancer that may regulate expression in specific spinal cells.

Genome-wide association studies of adolescent idiopathic scoliosis suggest candidate susceptibility genes.

Adolescent idiopathic scoliosis (AIS) is an unexplained and common spinal deformity seen in otherwise healthy children. Its pathophysiology is poorly understood despite intensive investigation. Although genetic underpinnings are clear, replicated susceptibility loci that could provide insight into etiology have not been forthcoming. To address these issues, we performed genome-wide association studies (GWAS) of ∼327 000 single nucleotide polymorphisms (SNPs) in 419 AIS families. We found strongest evidence of association with chromosome 3p26.3 SNPs in the proximity of the CHL1 gene (P < 8 × 10(-8) for rs1400180). We genotyped additional chromosome 3p26.3 SNPs and tested replication in two follow-up case-control cohorts, obtaining strongest results when all three cohorts were combined (rs10510181 odds ratio = 1.49, 95% confidence interval = 1.29-1.73, P = 2.58 × 10(-8)), but these were not confirmed in a separate GWAS. CHL1 is of interest, as it encodes an axon guidance protein related to Robo3. Mutations in the Robo3 protein cause horizontal gaze palsy with progressive scoliosis (HGPPS), a rare disease marked by severe scoliosis. Other top associations in our GWAS were with SNPs in the DSCAM gene encoding an axon guidance protein in the same structural class with Chl1 and Robo3. We additionally found AIS associations with loci in CNTNAP2, supporting a previous study linking this gene with AIS. Cntnap2 is also of functional interest, as it interacts directly with L1 and Robo class proteins and participates in axon pathfinding. Our results suggest the relevance of axon guidance pathways in AIS susceptibility, although these findings require further study, particularly given the apparent genetic heterogeneity in this disease.