A full genome screening in a large Tunisian family affected with thyroid autoimmune disorders

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1 (2001) 2, Nature Publishing Group All rights reserved /01 $ A full genome screening in a large Tunisian family affected with thyroid autoimmune disorders A Maalej 1, H Makni 1, F Ayadi 2, M Bellassoued 3, J Jouida 4, N Bouguacha 1, M Abid 3 and H Ayadi 1 1 Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, 3018 Sfax, Tunisie; 2 Service de Biochimie. EPS Habib Bourguiba, 3018 Sfax, Tunisie; 3 Service d Endocrinologie EPS Hedi Chaker, 3018 Sfax, Tunisie; 4 Dispensaire de Bir EI Hfai, Sidi Bouzid, Tunisie The autoimmune thyroid diseases (AITDs) including Graves disease (GD) and Hashimoto s thyroiditis (HT) are inherited as complex traits. We initiated a whole genome linkage study of patients with AITD, in order to identify the susceptibility genes involved in their pathogenesis. We studied 39 patients affected with GD or HT and 68 related controls, who belonged to a large consanguinous family composed of more than 200 members. Linkage analysis was performed using the lod score method under two arbitrary models, one dominant and one recessive. A positive lod score was found for D2S171, assuming a recessive mode of inheritance and 50% penetrance, which suggests the presence of a major AITD susceptibility gene on chromosome 2p21. However, no linkage was found with microsatellite markers spanning the HLA system. This locus localised outside MHC will be of interest for investigation of other autoimmune disorders. Genes and Immunity (2001) 2, Keywords: linkage study; autoimmune thyroid disorders; genome screening; susceptibility gene Introduction The autoimmune thyroid diseases (AITDs) include two related disorders, Graves disease (GD) and Hashimoto s thyroiditis (HT). Both diseases are characterized by a disturbance of function of the thyroid organ. GD is a form of hyperthyroidism characterized by goiter, ophtalmopathy, anti-thyroid stimulating hormone receptor (TSH-R) antibodies, classified as thyroid-stimulating and thyrotropinbinding inhibitory. There is a decrease of TSH and an increase of FT4 levels. HT is characterized by lymphocytosis, cytolysis, dense fibrosis, anti-thyroid plus antithyroperoxidase (anti-tpo) and anti-thyroglobuline (anti-tg) antibodies, and an increase of TSH and decrease of FT4 levels. These two forms of AITDs may be associated with other specific and nonspecific autoimmune diseases. AITDs is 10 times more frequent in women than in men 1 and occurs especially at 30 to 50 years of age, but may be seen in any age group, including children. The pathogenesis of the AITDs involves a complex interaction between genetic and environmental factors. 2 4 Indeed, epidemiological evidence for a genetic predisposition to AITDs is exhibited by clustering in families, 5 giving a sibling risk ratio ( s ) of 15, 6 and a high concordance rate in monozygotic twins compared to dizygotic twins. 7,8 Several candidate genes have been examined for a possible contribution to genetic susceptibility to the Correspondence: Hammadi Ayadi, Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine, avenue Majida Boulila 3018, Sfax, Tunisie. hammadi.ayadi fmsf.rnu.tn This work was supported by the Délégation Générale de la Recherche Scientifique et Technique (DGRST, TUNISIA). Received 2 August 2000; revised 6 December 2000; accepted 19 December 2000 AITDs, by means of both association and linkage analyses. These have included human leukocyte antigen (HLA), 9,10 as well as immunoglobulin heavy chain genes, 11 T cell receptor, 12,13 thyroid stimulating hormone receptor, 14 thyroid peroxidase, 15 and cytotoxic T lymphocyte associated-4 (CTLA-4) genes. However, with the exception of HLA, which showed evidence of association but not linkage in some studies, 16 all other candidate genes examined gave negative or equivocal results. Recently, five potential susceptibility loci for thyroid autoimmune disorders have been mapped to chromosomes 14q31, 17 Xq21.33, 18 20q11.2, 19 2q33, 20 and 18q However, no susceptibility gene for AITDs susceptibility has been identified. We report a genome-wide search using 214 microsatellite markers with an average spacing of 25 cm in a large consanguinous Tunisian family. Parametric linkage analysis revealed linkage to chromosome 2p21. The maximum pairwise lod score obtained after correction for multiple testing was equal to 3.00 with the microsatellite marker D2S171, and lod scores 1 were obtained for six additional markers. Results Genome screening for linkage using the set of 214 microsatellite markers showed evidence of linkage at a single marker located on chromosome 2, D2S171, with a MMLS-c score of 3.03 (Table 1). Six additional markers gave MMLS-c 1.0: D2S174 (1.17), D3S1276 (1.40), D3S1279 (1.44), D4S1597 (1.78), D10S249 (1.38) and D22S274 (1.01) (Table 1). A total of 36 additional microsatellite markers, including 15 markers located to a 56 cm region flanking D2S171 were used in the extended panel of individuals, and linkage analysis was perfor-

2 72 Table 1 Positive lod scores obtained from the genome screening performed on the AKR family Marker locus z (model A) z (model B) MMLS-c D2S D2S D3S D3S D4S D10S D22S *z (model A), parametric lod score under dominant mode of inheritance; z (model B), parametric lod score under recessive mode of inheritance; MMLS-c, maximum lod score corrected. Table 2 Detailed chromosome 2 analysis Marker Map position z z MMLS-c locus (cm) (model A) (model B) D2S D2S D2S D2S D2S D2S D2S D2S D2S D2S D2S D2S D2S D2S D2S med. As shown in Table 2, D2S171 gave similar evidence of linkage in the extended family (MMLS-c = 3.00), although weak or no evidence of linkage was found at flanking markers. Duplicate genotyping was performed at D2S171 in order to eliminate the possibility of a positive result due to inaccurate genotype determination, and the results were consistent with the first results obtained for the group studied. The analysis of six microsatellites over a 16 cm region around the MHC, three microsatellites over 16 cm around the CTLA-4 locus, four microsatellites over 20 cm encompassing the Ig VH genes complex and three microsatellites covering the C TCR locus did not show any linkage to these markers (MMLS-c 0). Discussion We have searched for a susceptibility gene for AITDs by microsatellites Genéthon panel and parametric lod score using a consanguinous Tunisian family living in a geographicaly isolated area. Our analysis showed suggestive evidence of linkage to one marker on chromosome 2p21 (D2S171), with a MMLS-c = 3.0, however, no evidence for linkage was found to these markers located in the vicinity of this marker. As we confirmed genotype data at D2S171 by duplicate typing, we propose that the most likely explanation for this observation was a combination of reduced efficiency of typing at flanking markers due to limited DNA availability and random effects due to variation in the informativity of subfamilies between different markers. The examination of genes mapped to this region showed that the hfkbp-12 gene which modulates the immune response was mapped near D2S171 and could be therefore a candidate gene in AITDs pathogenesis. 22 In addition, our study showed additional potential regions located on chromosomes 3 (D3S1276), (D3S1279), 10 (D10S249) and 22 (D22S274). None of the previously reported linkage studies on chromosome 14q31, 17 Xq , 18 20q11.2, 19 2q33, 20 and 18q21 21 were reproduced in our study. The lod score analysis involving the MHC, the CTLA-4, the TCR C and the Ig VH regions, allowed rejection of linkage of these markers to AITDs development. These data may be partly explained by the fact that we studied an isolated family with a very high consanguinity rate, suggesting the role of a major founder effect. The lack of linkage of the AITDs to the MHC region and CTLA-4 gene was in agreement with previous results, 16,23,24 and supported the notion that HLA and CTLA-4 genes could be considered only as minor contributors to the development of these diseases. Genetic association studies may be more appropriate to reveal such suceptibility genes. In summary, our data suggest that a gene locus conferring susceptibility to AITDs is located on chromosome 2p21. This locus will be of interest to explore in other autoimmune diseases as well as in other families with AITDs. Patients and methods Subjects Subjects were recruited from a single large family named Akr, with high prevalence of AITDs, consisting of four generations of more than 200 members, including 46 patients subdivided into 26 patients affected with GD and 20 patients affected with HT. 25 The female/male ratio was 0.73 in GD patients and 5.66 in HT patients (Figure 1). Patient ages at the onset of the disease ranged from 12 to 58 years with a mean age of 33.2 years. The diagnosis of GD was based on the presence of biochemical hyperthyroidism, as indicated by a decrease of TSH, an increase of T4 levels and positive TSH receptor antibody, in association with diffuse goiter or the presence of exophthalmos. The diagnosis of HT was based on the presence of thyroid hormone-replaced primary hypothyroidism as defined as a TSH level above the upper limits associated with positive titers of thyroid autoantibodies (anti-thyroid peroxidase or anti-thyroglobulin) and a palpable goiter. Methods Genotyping: Genomic DNA was extracted from 10 ml of peripheral blood lymphocyte (PBL) of Akr patients, healthy Akr-related controls and normal subjects derived from the same area using standard methods. 26 Akr patients and controls were genotyped with 214 highly polymorphic markers separated by a mean distance of 25 cm. Genotyping was undertaken using a radioactive detection system. Polymorphic microsatellites were amplified

3 73 Figure 1 Akr pedigree. ( ) Graves disease patient; ( * *) Hashimoto s thyroiditis patient; (D) affected subject; (TD) control subject; ( B ) consanguinous marriage.

4 74 in 96-well Falcon flexi plates by the polymerase chain reaction (PCR) using Techne thermocyclers. PCRs were performed in a final volume of 50 l, containing 40 ng of genomic DNA, 50 pmol of each primer, 1.25 mm dntps, 10 mm Tris ph 9, 50 mm KCI, 1.5 mm MgCl 2 0.1% Triton X-100, 0.01% gelatin and 1 unit of Taq DNA polymerase. Each PCR was performed using a hot-start procedure, and amplification was carried out using 35 cycles of denaturation at 94 for 40 s, with annealing at 55 for 30 s, followed by a final elongation step at 72 C for 2 min. The reaction products were pooled into size-specific groups and aliquots were denaturated at 96 for 2 min. A 5- l aliquot of each sample was separated by size on a 6% denaturing polyacrylamide gel with 8.3 m urea. The separated bands were transferred onto nylon membrane (Hybond N + ) and visualized by autoradiography, after exposition to Kodak XAR-5 film for 2 48 h at 70 C. Linkage analysis: Genome-wide screening for linkage to AITDs was performed on a subset of 92 individuals. Twenty unrelated control individuals, living in the same geographic area, were also genotyped for these markers, and allele frequencies were estimated for the controls and unrelated individuals in the Akr family. Linkage analysis was performed using the LINKAGE package, 27 under two arbitrary models, one dominant (disease allele frequency: 0.01; penetrance in heterozygotes and homozygotes: 0.5; phenocopy rate: 0.0), and one recessive (disease allele frequency: 0.01; penetrance in homozygotes: 0.5; phenocopy rate: 0.0), for a recombination fraction = 0.0. For each marker, a corrected parametric linkage score depending on these two results (MMLS-c) was obtained as the maximum of both lod score values, reduced by 0.30 according to a method proposed by Greenberg et al. 28 Fine exploration of chromosome 2 region was performed on a total of 107 individuals, including the first set of 92 individuals used for genome-wide screening. Linkage analysis was performed, using the same statistics as for the genome screening. Because of the complexity of this family characterized by the existence of many consanguinity loops and owing to computational efficiency, the Akr family used for genome screening was simplified into sub-families. Acknowledgements We thank Dr Cécile Julier for computer assistance and critical reading of the manuscript, Dr Christine Petit for her interest and support, and A Boulila-Elgaied and A Mziou for their technical assistance. References 1 Tunbridge WMG, Evered DC, Hall R et al. 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