The aim of this study is to clarify the implication of ciliary pathway on the onset of the spinal curvature that occurs in Adolescent Idiopathic Scoliosis (AIS) patients through functional studies of two genes: POC5 and TTLL11. Since the genetic implication for AIS is accepted, many association and candidate gene analysis revealed the implication of ciliary genes.

The characterisation of these two proteins was assessed by qPCR, WB and immunofluorescence in vitro using control cells and cells derived from AIS patients. The impact of genetic modification of these genes on the functionality of the proteins in vitro and in vivo was analysed in zebrafish model created by CRISPR/Cas9 using microCT and histologic analysis.

Our study revealed that mutant cells, for both gene, were less ciliated and the primary cilia was significantly shorter compared to control cells. We also observed a default in cilia glutamylation by immunofluorescence and Western Blot. Moreover, we observed in both zebrafish model, a 3D spine curvature similar to the spinal deformation in AIS. Interestingly, our preliminary results of immunohistology showed a retinal defect, especially at the cone cell layer level.

This study strongly supports the implication of the ciliary pathway in the onset of AIS and this is the first time that a mechanism is described for AIS. Indeed, we show that shorter cilia could be less sensitive to environmental factors due to lower glutamylation and result in altered signalling pathway. Identifying the biological mechanism involved is crucial for elucidating AIS pathogenesis.

Introduction

Marfan syndrome (MFS) is a common connective tissue disorder affecting one in 3300 people worldwide, and is caused by unique mutations in the 65 exon gene for fibrillin-1—an essential microfibril component of ligaments, tendons, and muscle. A recently discovered feature in the Marfan mouse model is increased concentrations of transforming growth factor β, resulting in overgrowth. 70% of patients with MFS have scoliosis of some degree. Can lessons be learned from MFS aetiology and treatment that apply to idiopathic adolescent scoliosis? We aimed to establish whether there is a relationship between the type and location of mutation, and the presence and degree of severity of scoliosis, in patients with MFS.

Introduction: Adolescent idiopathic scoliosis (AIS) is described as a sex-influenced autosomal dominantly inherited disorder with females more often affected than males, and operative ratio of 7F:1M (Child et al. 1999). Two AIS loci have been reported on chromosome 17p11 (Salehi et al. 2002) and chromosome 19p13.3 (Chan et al. 2002) in the Italian and Chinese populations, respectively. Three other susceptibility AIS loci on chromosome 6q, distal 10q and 18q (Wise et al. 2000), and more recently primary candidate regions on chromosomes 6, 9, 16, and 17 (Miller et al, 2005) have also been reported.

Purpose: o perform a genome scan for suitable UK multiplex families and identify new genetic loci for AIS.

Method: NA samples from 208 subjects (134 affected, 17 reduced penetrance members and 79 normal) from 25 multi-generation British families with confirmed diagnosis of AIS were selected from our AIS family database, and genotyped for 410 polymorphic markers from the entire genome, spaced at 10 cM intervals. Genotypic data were exported into Cyrillic to construct the most likely inherited haplotypes for each chromosome and in each family. Two–point LOD scores were calculated using MLINK initially for the entire genotypic data, and again for the affected meioses only, followed by GENEHUNTER for multipoint linkage analysis for each family.

Results: Overall, 170 560 genotypes were obtained and analysed. DNA samples from 250 subjects from the 25 families are currently available for further genotyping and saturation mapping. Our AIS families show absence of linkage to the X chromosome as well as previously reported AIS loci, except for chromosome 9q and 17q as reported by Miller et al. (2005). Preliminary inspection of inherited haplotypes indicates that a number of these families may be segregating with several new AIS loci with LOD scores ranging from 1.0 – 3.63 for various DNA markers on 15 different chromosomes. Linkage evaluation and comprehensive saturation mapping of the two loci with the highest LOD scores of 3.63 and 4.08 for chromosomes 9q and 17q respectively were conducted and these regions were successfully refined. Candidate genes are currently being screened.

Conclusion: Preliminary evidence already indicates genetic heterogeneity of AIS. Candidate genes from the highest LOD score regions are at present being screened.

Purpose: To perform a genome scan for suitable UK multiplex families and identify new genetic loci for AIS.

Method: DNA samples from 208 subjects (134 affected, 17 reduced penetrance members and 79 normal) from 25 multi-generation British families with confirmed diagnosis of AIS were selected from our AIS family database, and genotyped for 410 polymorphic markers from the entire genome, spaced at 10 cM intervals. Genotypic data were exported into Cyrillic to construct the most likely inherited haplotypes for each chromosome and in each family. Two–point LOD scores were calculated using MLINK initially for the entire genotypic data, and again for the affected meioses only, followed by GENEHUNTER for multipoint linkage analysis for each family.

Results: Overall, 170,560 genotypes were obtained and analysed. DNA samples from 250 subjects from the 25 families are currently available for further genotyping and saturation mapping. Preliminary inspection of inherited haplotypes indicates that a number of these families may be segregating with several new AIS loci with LOD scores ranging from 1.0 – 3.6 for various DNA markers on 15 different chromosomes (1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 13, 16, 17, 20, 21), and absence of linkage to the X chromosome. Linkage evaluation and comprehensive saturation mapping of the 2 loci with the highest LOD scores were conducted and these regions were successfully refined. Candidate genes are currently being screened.

Conclusion: Preliminary evidence already indicates genetic heterogeneity of AIS. Candidate genes from the two highest LOD score loci are at present being screened.

Introduction: AIS is described as a sex-influenced auto-somal dominantly inherited disorder with females more often affected than males (operative ratio 7F:1M) 1. Two AIS loci have been reported on chromosomes 17p112 and 19p13.33 in the Italian and Chinese populations, respectively. Other susceptibility AIS loci on chromosomes 6p, distal 10q and 18p4, and more recently to chromosomes 6, 9, 16, and 175, and 19p136 have also been reported, in the American population.

Purpose: To perform a genome scan for suitable UK multi-generation families and identify new genetic loci for AIS.

Method: DNA samples from 208 subjects (116 affected members) from 25 British families with confirmed diagnosis of AIS were selected from our family database, and genotyped for 410 polymorphic markers from the entire genome, spaced at ~10 cM intervals. Using Cyrillic, most likely inherited haplotypes were constructed for each chromosome and family. Statistical analyses were calculated using MLINK and GENEHUNTER, initially for the entire genotypic data, and again for affected meioses only.

Results: 170,560 genotypes were obtained and analysed. Our AIS families show no linkage to the X chromosome. Preliminary inspection of inherited haplotypes indicates a number of families may be segregating with several new AIS loci with LOD scores from 1.0–3.64 for markers on 15 different chromosomes. Linkage analysis and saturation mapping of the 2 highest LOD score regions on chromosomes 9q34 and 17q25 were conducted. These regions were successfully refined and candidate genes are being screened.

Conclusion: Preliminary evidence already indicates genetic heterogeneity of AIS. Candidate genes from the two highest LOD score loci are at present being screened.