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.

ptk7 mutant zebrafish models of congenital and idiopathic scoliosis implicate dysregulated Wnt signalling in disease.

Scoliosis is a complex genetic disorder of the musculoskeletal system, characterized by three-dimensional rotation of the spine. Curvatures caused by malformed vertebrae (congenital scoliosis (CS)) are apparent at birth. Spinal curvatures with no underlying vertebral abnormality (idiopathic scoliosis (IS)) most commonly manifest during adolescence. The genetic and biological mechanisms responsible for IS remain poorly understood due largely to limited experimental models. Here we describe zygotic ptk7 (Zptk7) mutant zebrafish, deficient in a critical regulator of Wnt signalling, as the first genetically defined developmental model of IS. We identify a novel sequence variant within a single IS patient that disrupts PTK7 function, consistent with a role for dysregulated Wnt activity in disease pathogenesis. Furthermore, we demonstrate that embryonic loss-of-gene function in maternal-zygotic ptk7 mutants (MZptk7) leads to vertebral anomalies associated with CS. Our data suggest novel molecular origins of, and genetic links between, congenital and idiopathic forms of disease.

Genomic Analyses of Patients With Unexplained Early-Onset Scoliosis.

STUDY DESIGN: To test for rare genetic mutations, a cohort of patients with unexplained early-onset scoliosis (EOS) was screened using high-density microarray genotyping. A cohort of patients with adolescent idiopathic scoliosis (AIS) was similarly screened and the results were compared. SUMMARY OF BACKGROUND DATA: Patients with scoliosis in infancy or early childhood (EOS) are at high risk for progressive deformity and associated problems including respiratory compromise. Early-onset scoliosis is frequently associated with genetic disorders but many patients present with nonspecific clinical features and without an associated diagnosis. The authors hypothesized that EOS in these patients may be caused by rare genetic mutations detectable by next-generation genomic methods. METHODS: The researchers identified 24 patients with unexplained EOS from pediatric orthopedic clinics. They genotyped them, along with 39 connecting family members, using the Illumina OmniExpress-12, version 1.0 beadchip. Resulting genotypes were analyzed for chromosomal changes, specifically copy number variation and absence of heterozygosity. They screened 482 adolescent idiopathic scoliosis (AIS) patients and 744 healthy controls, who were similarly genotyped with the same beadchip, for chromosomal changes identified in the EOS cohort. RESULTS: Copy number variation and absence of heterozygosity analyses revealed a genetic diagnosis of chromosome 15q24 microdeletion syndrome in 1 patient and maternal uniparental disomy of chromosome 14 in a second one. Prior genetic testing and clinical evaluations had been negative in both cases. A large novel chromosome 10 deletion was likely causal in a third EOS patient. These mutations identified in the EOS patients were absent in AIS patients and controls, and thus were not associated with AIS or found in asymptomatic individuals. CONCLUSIONS: These data underscore the usefulness of updated genetic evaluations including high-density microarray-based genotyping and other next-generation methods in patients with unexplained EOS, even when prior genetic studies were negative. These data also suggest the intriguing possibility that other mutations detectable by whole genome sequencing, as well as epigenetic effects, await discovery in the EOS population.

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.

Clinical, Molecular, and Genetic Characteristics of PAPA Syndrome: A Review.

PAPA syndrome (Pyogenic Arthritis, Pyoderma gangrenosum, and Acne) is an autosomal dominant, hereditary auto-inflammatory disease arising from mutations in the PSTPIP1/CD2BP1 gene on chromosome 15q. These mutations produce a hyper-phosphorylated PSTPIP1 protein and alter its participation in activation of the "inflammasome" involved in interleukin-1 (IL-1ß) production. Overproduction of IL-1ß is a clear molecular feature of PAPA syndrome. Ongoing research is implicating other biochemical pathways that may be relevant to the distinct pyogenic inflammation of the skin and joints characteristic of this disease. This review summarizes the recent and rapidly accumulating knowledge on these molecular aspects of PAPA syndrome and related disorders.

Understanding genetic factors in idiopathic scoliosis, a complex disease of childhood.

Idiopathic scoliosis (AIS) is the most common pediatric spinal deformity, affecting ~3% of children worldwide. AIS significantly impacts national health in the U. S. alone, creating disfigurement and disability for over 10% of patients and costing billions of dollars annually for treatment. Despite many investigations, the underlying etiology of IS is poorly understood. Twin studies and observations of familial aggregation reveal significant genetic contributions to IS. Several features of the disease including potentially strong genetic effects, the early onset of disease, and standardized diagnostic criteria make IS ideal for genomic approaches to finding risk factors. Here we comprehensively review the genetic contributions to IS and compare those findings to other well-described complex diseases such as Crohn's disease, type 1 diabetes, psoriasis, and rheumatoid arthritis. We also summarize candidate gene studies and evaluate them in the context of possible disease aetiology. Finally, we provide study designs that apply emerging genomic technologies to this disease. Existing genetic data provide testable hypotheses regarding IS etiology, and also provide proof of principle for applying high-density genome-wide methods to finding susceptibility genes and disease modifiers.

Dual role of the TRPV4 channel as a sensor of flow and osmolality in renal epithelial cells.

Gain/loss of function studies were utilized to assess the potential role of the endogenous vanilloid receptor TRPV4 as a sensor of flow and osmolality in M-1 collecting duct cells (CCD). TRPV4 mRNA and protein were detectable in M-1 cells and stably transfected HEK-293 cells, where the protein occurred as a glycosylated doublet on Western blots. Immunofluorescence imaging demonstrated expression of TRPV4 at the cell membranes of TRPV4-transfected HEK and M-1 cells and at the luminal membrane of mouse kidney CCD. By using intracellular calcium imaging techniques, calcium influx was monitored in cells grown on coverslips. Application of known activators of TRPV4, including 4alpha-PDD and hypotonic medium, induced strong calcium influx in M-1 cells and TRPV4-transfected HEK-293 cells but not in nontransfected cells. Applying increased flow/shear stress in a parallel plate chamber induced calcium influx in both M-1 and TRPV4-transfected HEK cells but not in nontransfected HEK cells. Furthermore, in loss-of-function studies employing small interference (si)RNA knockdown techniques, transfection of both M-1 and TRPV4-transfected HEK cells with siRNA specific for TRPV4, but not an inappropriate siRNA, led to a time-dependent decrease in TRPV4 expression that was accompanied by a loss of stimuli-induced calcium influx to flow and hypotonicity. It is concluded that TRPV4 displays a mechanosensitive nature with activation properties consistent with a molecular sensor of both fluid flow (or shear stress) and osmolality, or a component of a sensor complex, in flow-sensitive renal CCD.

CHD7 gene polymorphisms are associated with susceptibility to idiopathic scoliosis.

Idiopathic scoliosis (IS) is the most common spinal deformity in children, and its etiology is unknown. To refine the search for genes underlying IS susceptibility, we ascertained a new cohort of 52 families and conducted a follow-up study of genomewide scans that produced evidence of linkage and association with 8q12 loci (multipoint LOD 2.77; P=.0028). Further fine mapping in the region revealed significant evidence of disease-associated haplotypes (P<1.0 x 10-4) centering over exons 2-4 of the CHD7 gene associated with the CHARGE (coloboma of the eye, heart defects, atresia of the choanae, retardation of growth and/or development, genital and/or urinary abnormalities, and ear abnormalities and deafness) syndrome of multiple developmental anomalies. Resequencing CHD7 exons and conserved intronic sequence blocks excluded coding changes but revealed at least one potentially functional polymorphism that is overtransmitted (P=.005) to affected offspring and predicts disruption of a caudal-type (cdx) transcription-factor binding site. Our results identify the first gene associated with IS susceptibility and suggest etiological overlap between the rare, early-onset CHARGE syndrome and common, later-onset IS.

Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathways.

The TRPV4 calcium-permeable channel was cloned from mouse kidney M-1 cells, and the effect of temperature modulation on channel gating/activation by physical and chemical signals was evaluated. A TRPV4 cDNA construct with a C-terminal V5 epitope was stably transfected into human embryonic kidney (HEK) 293 and Chinese hamster ovary cells resulting in high levels of expression at the plasma membrane. Channel activation was assessed from changes in calcium influx (fura-2 fluorescence measurements) or whole cell currents (patch clamp analysis). At room temperature (22-24 degrees C), exposure of TRPV4-transfected cells to hypotonic medium (225 mOsm/liter) or a non-protein kinase C (PKC)-activating phorbol ester derivative, 4alpha-phorbol 12,13-decanoate (100 nm), induces modest channel activation, whereas phorbol 12-myristate 13-acetate (100 nm), a PKC-activating phorbol ester, and shear stress (3-20 dyne/cm2) had minimal or no effect on channel activation. In contrast, at elevated temperatures (37 degrees C) the channel was rapidly activated by all stimuli. Inhibition of PKC by calphostin C (50 nm) or staurosporine (500 nm) abolished phorbol 12-myristate 13-acetate-induced activation of the channel without affecting the response to other stimuli. Ruthenium red (1 microm) effectively blocked the channel activity by all stimuli. It is concluded that temperature is a critical modulator of TRPV4 channel gating, leading to activation of the channel by a diverse range of microenvironmental chemical and physical signals utilizing a least two transduction pathways, one PKC-dependent and one PKC-independent. The convergence of multiple signals and transduction pathways on the same channel indicate that the channel functions as a molecular integrator of microenvironmental chemical and physical signals.