Association of an estrogen-sensitive Pax1-Col11a1-Mmp3 signaling axis with adolescent idiopathic scoliosis

Joint Public Review:

Summary: This study follows up on previous work showing a female-specific enhancer region of PAX1 is associated with adolescent idiopathic scoliosis (AIS). This new analysis combines human GWAS analysis from multiple countries to identify a new AIS-associated coding variant in the COL11A1 gene. Two nonsynonymous variants were found to be significantly associated with AIS: MMP14 p.Asp273Asn and COL11A1 p.Pro1335Leu, the latter of which had the more robust association and remained significant when females were tested independent of males. Using a Pax1 knockout mouse they go on to find that PAX1 and Collagen XI protein are expressed in the intervertebral discs (IVDs) and robustly in the growth plate, showing that COL11A1 expression is reduced in Pax1 mutant growth plate. Moreover, other AIS-associated genes, Gpr126 and Sox6, were also reduced in Pax1 mutant mice, suggesting a common pathway is involved in AIS. The proposed implication of a Pax1-Col11a1-Mmp3 signaling axis modulated by estrogen signaling suggests a potential mechanism by which young women have more severe scoliosis than young men, as is observed in humans.

Strengths: This work integrates a large cohort of human genetic data from AIS patients and controls from diverse ethnic backgrounds, across the globe. This work attempts to functionally test their findings in vivo and by use of cell culture. The authors propose an interesting model which warrants in depth investigation.

Weaknesses: There are concerns regarding the candidacy of COL11A1 p.Pro1335Leu that need to be addressed and clarified. Many of the main functional work was done in cell culture and not in vivo. Moreover, the evidence linking COL11A1 p.Pro1153Leu to AIS is indirect, making unclear whether impaired COL11A1 function can cause scoliosis in the mouse model, thus diminishing the strength of the conclusions regarding the proposed pathogenicity of COL11A1 p.Pro1335Leu.

Author Response

We thank the reviewers for truly valuable advice and comments. We have made multiple corrections and revisions to the original pre-print accordingly. Here we address 2 major points.

1. Regarding the genetic association of the common COL11A1 variant rs3753841 (p.(Pro1335Leu)), we do not propose that it is the sole risk variant contributing to the association signal we detected and have clarified this in the manuscript. We concluded that it was worthy of functional testing for reasons described here. Although there were several common variants in the discovery GWAS within and around COL11A1, none were significantly associated with AIS and none were in linkage disequilibrium (R2>0.6) with the top SNP rs3753841. We next reviewed rare (MAF<=0.01) coding variants within the COL11A1 LD region of the associated SNP (rs3753841) in 625 available exomes representing 46% of the 1,358 cases from the discovery cohort. The LD block was defined using Haploview based on the 1KG_CEU population. Within the ~41 KB LD region (chr1:103365089- 103406616, GRCh37) we found three rare missense mutations in 6 unrelated individuals, Author response table 1. Two of them (NM_080629.2: c.G4093A:p.A1365T; NM_080629.2:c.G3394A:p.G1132S), from two individuals, are predicted to be deleterious based on CADD and GERP scores and are plausible AIS risk candidates. At this rate we could expect to find only 4-5 individuals with linked rare coding variants in the total cohort of 1,358 which collectively are unlikely to explain the overall association signal we detected. Of course, there also could be deep intronic variants contributing to the association that we would not detect by our methods. However, given this scenario, the relatively high predicted deleteriousness of rs3753841 (CADD= 25.7; GERP=5.75), and its occurrence in a Gly-X-Y triplet repeat, we hypothesized that this variant itself could be a risk allele worthy of further investigation.

Author response table 1.

We also appreciate the reviewer’s suggestion to perform a rare variant burden analysis of COL11A1. We conducted pilot gene-based analysis in 4534 European ancestry exomes including 797 of our own AIS cases and 3737 controls and tested the burden of rare variants in COL11A1. SKATO P value was not significant (COL11A1_P=0.18) but this could due to lack of power and/or background from rare benign variants that could be screened out using the functional testing we have developed.

1. Regarding functional testing, by knockdown/knockout cell culture experiments, we showed for the first time that Col11a1 negatively regulates Mmp3 expression in cartilage chondrocytes, an AIS-relevant tissue. We then tested the effect of overexpressing the human wt or variant COL11A1 by lentiviral transduction in SV40-transformed chondrocyte cultures. We deleted endogenous mouse Col11a1 by Cre recombination to remove the background of its strong suppressive effects on Mmp3 expression. We acknowledge that Col11a1 missense mutations could confer gain of function or dominant negative effects that would not be revealed in this assay. However as indicated in our original manuscript we have noted that spinal deformity is described in the cho/cho mouse, a Col11a1 loss of function mutant. We also note the recent publication by Rebello et al. showing that missense mutations in Col11a2 associated with congenital scoliosis fail to rescue a vertebral malformation phenotype in a zebrafish col11a2 KO line. Although the connection between AIS and vertebral malformations is not altogether clear, we surmise that loss of the components of collagen type XI disrupt spinal development. in vivo experiments in vertebrate model systems are needed to fully establish the consequences and genetic mechanisms by which COL11A1 variants contribute to an AIS phenotype.