Identification of ERα gene polymorphism in adolescent idiopathic scoliosis (AIS) L.-O. Lévesque, A. Moreau, PhD S. Leclerc, PhD, H. Labelle, MD, G. Grimard, MD, F. Moldovan, PhD.

What is adolescent idiopathic scoliosis ? 80% of scoliosis are idiopathic (without cause) Affects 2-3% of adolescents in Quebec Only 0,2% have a cobb angle over 40°

Do estrogens contribute to AIS? AIS affects mainly girls AIS appears at the same time that estrogens rise in young girls blood. Scoliosis progression can be predicted by bone age, menarche and pubertal stage, all of which are related to the presence of estrogens Environmental factors: phytoestrogens (nutrition)

Known receptors of estrogens There are 2 types of known receptors to the estrogens: ERα and ERβ Both are nuclear receptors of the steroids receptors super-family The ligand–receptor complex binds to ERE and AP-1 promoter sequence New evidences for a non-nuclear rapid action of the Estrogens The role of ERα and ERβ remains to be ascertained

Methodology All these experiments were done on human tissues or cell lines of human origin. MG-63, Control tissues and Scoliotic tissues all of which have been collected according to Sainte-Justine hospital’s ethic protocol By RT-PCR, we have seek ERα and ERβ in different human cell lines and scoliotic cell lines We have performed digestion by restriction enzymes on PCR amplicon to ascertain the presence of polymorphisms associated with ERα.

RFLPolymorphism Allele A Allele B Amplicons No enzymes PvuII XbaI Digestion enzyme enzyme Digested fragments

Results # of Scoliosis XbaI PvuII Type of Scoliosis Type of Curve sex 1er Cobb Cobb pre-op Patient 1 Xx Pp AIS Right thoracic F n/a 60 Patient 2 XX PP M 30 38 Patient 3 Paralytic Scoliosis Left thoracic 61 Patient 4 51 57 Patient 5 55 59 Patient 6 50 Patient 7 58

Expression of ERα and ERβ in human cells and scoliotic tissues by RT-PCR chondrcytes GAPDH ERα ERβ Patient A GAPDH ERα ERβ Patient B GAPDH ERα ERβ MG-63 GAPDH ERα ERβ

Relevance of this RFLP study Statistically related to curve progression in Japanese population (right thoracic) (Inoue et al. 1996) These RFLP are located in an intron Will not be expressed in the a.a. sequence May affect transcription factors binding RFX1 for example which also binds to growth factors promoters May affect the splicing Alternative splicing… Ex.: 66kDa and 46kDa

Conclusion & Perspectives A lot is left to investigate… ERα and ERβ was detected in osteoblasts but… What modulates them remains to be ascertain The full impact of those receptors on the scoliotic bone is still unclear The nuclear action of Estrogens has been well studied but there are evidence of non-nuclear action that should be investigated Detected the polymorphism in our population. Only on a small scale Correlate the presence of the polymorphism and ERα levels