LACK OF ASSOCIATION BETWEEN THE AGGRECAN GENE AND FAMILIAL IDIOPATHIC SCOLIOSIS +*Marosy, B; *Nzegwu, N; *Kumar, K; **Justice, CM; **Wilson, AF; *Miller, NH +Johns Hopkins University, School of Medicine, Baltimore, MD bmarosy@jhmi.edu Introduction: Familial idiopathic scoliosis (FIS) is a structural curvature of the spine whose genetic determinants and pattern of inheritance are not clear. The overall goal of the study is to define the genes responsible for this disorder utilizing a large, well- defined population. Data from a genomic screen of this large population combined with the current literature has identified an area on chromosome 15 of critical interest. This region includes aggrecan, a potential candidate gene for FIS. Aggrecan, a major component of cartilage has been linked to multiple skeletal disorders, and is known to play a role in human disc degeneration and osteoarthritis. A variable nucleotide tandem repeat (VNTR) polymorphism within exon 12 of the aggrecan gene has been identified within a region which encodes for the core protein containing numerous chondroitin sulfate attachment sites [Doege et al, 1997]. These sites play an important role in the water holding characteristics of cartilage found in joints and the processing of aggrecan. Variation of this polymorphism results in different lengths of core protein, which could potentially alter the biological function of the protein. A recent study associated this aggrecan polymorphism with curve severity in individuals with scoliosis [Merola et al, 2003]. The current work is directed to analyzing families with FIS and the aggrecan gene polymorphism. Materials and Methods: Families with two or more affected individuals identified through evidence of a ten-degree saggital curvature (Cobb angle) on standing anterioposterior radiographs were recruited with IRB approval. Blood samples were obtained from all individuals and DNA was extracted using standard protocols. The study population of families with FIS (202 families; 1198 individuals) underwent a genomic screen by the Center for Inherited Disease Research utilizing a standard automated fluorescent marker set (Weber 9 Screening Set). Families were stratified based on potential mode of inheritance. Model-independent sib-pair linkage analyses for both quantitative and discrete traits implemented in SIBPAL were utilized to screen each trait for evidence of linkage to each genetic marker. Model- dependant two-point linkage analysis was performed using Mlink from the LINKAGE software package. Quantitative and qualitative analysis of the genomic screen yielded significant results in the sample of families most likely to be X-linked dominant (XLD) (59 families, 354 individuals), marker d15s966 (p=0.00075). This sample of families was used for analysis of the aggrecan exon 12 polymorphism. Primers surrounding the VNTR region within exon 12 of the aggrecan gene were used to amplify genomic DNA from the study sample. Products were analyzed for allele length on agarose gels. Results: Initial linkage analyses of a subgroup of families representing an XLD mode of inheritance revealed significant results on chromosome 15q25-26 (p=0.00075), an area inclusive of the aggrecan gene. Fifty- nine unrelated families with familial idiopathic scoliosis, consisting of 354 individuals (224=F/130=M), were genotyped for a specific VNTR polymorphism within exon 12 of the aggrecan gene. Of the 354 individuals, 194 are affected (170=F/24=M), 159 are unaffected (53=F/106=M), and the affectation status of 1 individual is unknown (1=F/0=M). Within this patient sample, 11 of the 13 known alleles were identified (see Table 1). Model-independent sib-pair linkage analysis was performed to identify linkage between scoliosis and the exon 12 polymorphism [SIBPAL, S.A.G.E.]. No linkage was found with p- values of 0.5 and 0.2, respectively, when analyzing the data as a quantitative and qualitative trait respectively. Tests of association were performed using ASSOC and TDT. ASSOC, a quantitative test found no significance (p=0.47) at the aggrecan locus. The TDT test was performed on families with all children, and then on families and only 1 affected child. No significance was found with p-values of 0.9 and 0.74, respectively. Table 1. Distribution of Allele Frequencies All Families Affected Individuals Unaffected Individuals Obs. Allele Repeat No. Freq n=670 % Freq n=364 % Freq n=304 % 33 0 0 0 0 0 0 32 8 1.2 4 1.1 4 1.3 29 69 10.3 37 10.1 32 10.5 28 379 56.5 207 57.8 170 55.9 27 147 21.9 78 21.4 69 22.7 26 20 3.0 9 2.5 11 3.6 25 2 0.3 1 0.3 1 0.3 22 24 3.5 14 3.8 10 3.3 21 9 1.3 6 1.6 3 1 20 0 0 0 0 0 0 19 2 0.3 2 0.5 0 0 18 3 0.4 1 0.3 2 0.7 13 7 1.0 5 1.4 2 0.7 Discussion: Multiple studies have indicated the genetic nature of scoliosis, although the mode of inheritance is unclear. Heterogeneity plays a confounding role in the complexity of the disorder and underlines the fact that multiple genes may play a role in FIS. Aggrecan polymorphisms and mutations have been associated with variation in the functional properties of cartilage and its role in cartilage disorders. Variation within the exon 12 region containing numerous attachment sites for chondroitin sulfate would potentially result in a compromise of these sites and ultimately the biological function of the protein. Statistical analyses for the potential association of the exon 12 polymorphism and the scoliosis phenotype showed no evidence of linkage within the large sample of families affected with FIS. Despite the negative association reported here, the role of aggrecan in the etiology of cartilage based disease processes remains in question. Further investigation of the gene and its potential association to FIS through alternative polymorphisms and/or other mutations is required. Cited References: Doege KJ, Coulter SN, Meek LM, Maslen K, Wood JG. A human-specific polymorphism in the coding region of the aggrecan gene. J Biol Chem 1997;23:13974-13979. Merola AA, Mathur S, Igobou S, Brkaric M, Vigna F, Paulino C, Kubec J, Haher TR, Espat N. Polymorphism of the aggrecan gene as a marker for adolescent idiopathic scoliosis. SRS 38 th Annual meeting Quebec Canada September 2003. 51st Annual Meeting of the Orthopaedic Research Society Poster No: 1299