Development and improvement of EDTSA database Objective 1

Section 1: Thymic expression

Section 2: General gene information

Section 3: References

To accomplish the next objectives (2 and 3), the last year an extensive repository of human thymic samples (n=192) have been collected and processed From most of thymic donors, serum and PBMCs have been obtained from peripheral blood

Genetic basis of inter- and intraindividual thymic expression variability Objective 2.1 TSHR: A candidate gene as the main autoantigen in Graves-Basedow disease

Haplotype definition and case-control study: - Samples included 360 gDNAs from: * 192 human thymic donors (see table before). * 137 human patients suffering Autoimmune Thyrotoxicosis (Graves-Basedow disease). * 31 human patients suffering a non-autoimmune thyroid disease (Multinodular goiter). - SNPs analyzed: 51 SNPs of TSHR gene have been selected and classified in two main sets: 1. Interesting SNPs mapping in the promoter region and those located in regions transcribed to mRNA. 2. TagSNPs capturing most of the genetic variability of the gene. TagSNPs have been selected using the Haploview software and the available data of HapMap project. - Genotyping system: SNPs genotyping is underway at the CRG (Centre for Genomic Regulation) by using the SNPlex TM System, that enables the simultaneous genotyping of up to 48 SNPs against a single biological sample. The date that all the results will be available to us is first week of december. - An interesting non-SNP polymorphism included in the analysis: An additional interesting polymorphism has been included in this analysis. It is a DIP (Deletion/Insertion Polymorphism) of 17 bp located in the early promoter of TSHR gene.

- Genotyping of DIP polymorphism: the 192 gDNAs from thymic donors and the 137 gDNAs from suffering Autoimmune Thyrotoxicosis have been genotyped for this polymorphism. del/ins del/del Genotyping by PCR Frequencies

-Association of DIP polymorphism with Autoimmune Thyrotoxicosis: Interestingly, the genotyping results indicate a statistically significant association of this DIP with Autoimmune Thyrotoxicosis. Using the dominant model, the presence of the insertion allele is associated with an increased susceptibility to the Autoimmune Thyrotoxicosis disease (p = 0.029, OR = 1.71).

Study of thymus from autoimmune patients Objective 2.2

We tested for the presence of common autoantibodies in sera samples from thymic donors. Autoantibodies tested were: - - Organ specific autoantibodies: TPO (Thyroid Peroxidase), TG (Thyroglobulin), TSHR (Thyroid Stimulating Hormone Receptor), ICA (Islet Cell Antibodies). - - Non-organ specific autoantibodies: AMA (Anti-Mitochondrial Antibodies), LKM (Liver/Kidney Microsomal Antibodies), ML (Smooth Muscle Antibodies), CPG (Gastric Parietal Cell Antibodies), ANA (Anti-Nuclear Antibodies). Ribosomal, dsDNA and ENAS (Extractable Nuclear Antigens) are only tested when ANA are positive above 1/320. To date, 75 sera have been tested or partially tested. Results are given in the table:

Analysis of interindividual variability of autoantigen expression in the thymus Objective 3

A set of 27 genes including autoantigens and specific cell marker genes have been selected. RNA from all samples (192 thymus) have been purified and cDNA will be obtained. qRT-PCR will be performed in the following groups: Among these samples there are 11 cases of Down Syndrome that will be subjected to additional studies in collaboration with Alex Marx group GroupsN Young thymi (< 10 years)83 Middle-age thymi (from 11 to 30 years)6 Adult thymi (from 31 to 60 years)39 Old thymi (> 61 years)60