A PAX1 Enhancer Locus Increases Risk of Idiopathic Scoliosis in Females. C. Wise1,2,3,4, S. Sharma1,13, D. Londono5, W. Eckalbar6,12, X. Gao1, I. Kou7, A. Takahashi7,8, M. Matsumoto9, J. A. Herring2,10, D. K. Burns11, S. Ikegawa7,8, N. Ahituv6,12, D. Gordon5 1) Seay Center for Musculoskeletal Research, Texas Scottish Rite Hosp, Dallas, TX; 2) Department of Orthopaedic Surgery, University of Texas Southwestern Medical Center at Dallas, Dallas, TX; 3) Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center at Dallas, Dallas, TX; 4) Department of Pediatrics, University of Texas Southwestern Medical Center at Dallas, Dallas, TX; 5) Department of Genetics and Human Genetics Institute, Rutgers University, Piscataway, NJ; 6) Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA; 7) Laboratory for Statistical Analysis, Center for Genomic Medicine, RIKEN, Tokyo, Japan; 8) Laboratory of Bone and Joint Diseases, Center for Genomic Medicine, RIKEN, Tokyo, Japan; 9) Department of Orthopaedic Surgery, School of Medicine, Keio University, Tokyo, Japan; 10) Department of Orthopaedics, Texas Scottish Rite Hospital for Children, Dallas, TX; 11) Department of Pathology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX; 12) Institute for Human Genetics, University of California San Francisco, San Francisco, CA; 13) School of Biotechnology, Shri Mata Vaishno Devi University, Katra, India.

   Idiopathic scoliosis (IS) is the most common pediatric spinal disorder, affecting more than thirty million children worldwide. IS exhibits a striking ten-fold greater risk of progressive disease in females for reasons that are unknown. Although IS is highly heritable, few replicated risk loci have been identified. To discover and characterize new IS genetic risk factors, we performed a two-stage GWAS that combined a prior family-based GWAS with a new case-control study (N= 3,102 individuals total). Our analyses identified an association with 20p11 SNPs (combined P=1.33x10-8) clustering ~186 kb distal to the 5 end of the PAX1 homeobox gene (OMIM #167411). Surprisingly, several of the 20p11 SNPs were previously associated with protection against early-onset male pattern baldness, suggesting that the region participates in sexually dimorphic gene expression. Accordingly, stratification by sex yielded IS association in females but not males for both GWAS stages (region combined P=6.89x10-9 in females and P=0.71 in males). We replicated the IS association with top SNP rs6137473 in an independent NHW female cohort (OR=1.67, P = 2.4x10-4), and in a Japanese female cohort (OR = 1.19, P =3.7x10-3). Combined female results across all four studies yielded P=2.15x10-10, OR=1.30 for this marker. Spontaneous mouse pax1 deletion mutants develop scoliosis and other spinal anomalies. Consequently we hypothesized that the Chr 20p11 IS susceptibility locus functions through cis-acting regulation of PAX1 expression. To identify potential enhancers in the region we selected highly conserved sequences and tested activity in zebrafish transgene assays, revealing two candidate enhancers. One enhancer, SE7, drove expression in somitic muscle and spinal cord and harbored top disease-associated SNP alleles that are predicted to disrupt transcription factor binding. Re-sequencing SE7 in IS cases revealed a common haplotype that when tested in zebrafish abolished the enhancer activity of this sequence. In addition, immunohistochemistry in mouse spinal tissues revealed most persistent Pax1 protein expression in myogenic cells beginning at E16.5 to day P84. This study is the first to yield insights into the well-known and puzzling sex bias in IS. Our data suggest that Chr 20p11 variants increase susceptibility to IS by altering cis-acting regulatory sequences that may participate in sexually dimorphic PAX1 expression in spinal muscle and/or nerve cells.

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