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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
age number of samples
1w-13w 27
14w-26w 10
27w-39w 10
40w-52w 4
1y-4y 25
5y-10y 7
11y-20y 5
21y-30y 1
31y-40y 3
41y-50y 10
51y-60y 26
61y-70y 33
71y-80y 24
81y-90y 3
unknown 4
total 192
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 SNPlexTM 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
/in
s
del
/in
s
del
/in
s
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
Groups N
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