Allele and haplotype frequencies of the Czech Graves´disease patients

We analyzed the HLA-<i>DRB1</i>, HLA-<i>DQA1</i> and HLA-<i>DQB1</i> allelic groups in 206 retrospectively observed Czech patients who had been diagnosed with Graves' disease according to hormonal profile (low/suppressed TSH with simultaneously elevated freeT4, freeT3, presence of thyrotropin receptor antibodies (TRAK)) as well as sonographic examination of the thyroid gland. Patients were recruited during the years 2017-2020 mainly from the outpatients of the Institute of Endocrinology in Prague. These patients are continuously monitored at the Institute, which gives us detailed long-term or even lifelong retrospective information on the status of their disease. Next generation sequencing performed on a MiSeq (Illumina) was used for haplotyping. In the three HLA genes (<i>DRB1</i>, <i>DQA1</i> and <i>DQB1</i>), exon 2 was amplified, since this part of the HLA molecule determines its antigenic properties. Amplification of DNA sequences was performed using certified kits (CE 0197, CE IVD) of allele-specific pre-coated PCR strips PROTRANS S4 HLA-DRB1 * (cat. No. 34 04), PROTRANS S3 HLA-DQB1 * (cat. No. 33 09) and PROTRANS S2 HLA-DQA1 * (cat. No. 32 08) from the manufacturer PentaGen, Protrans, Germany.

本研究对206例回顾性纳入的捷克格雷夫斯病（Graves' disease）患者的人类白细胞抗原（HLA）DRB1、DQA1及DQB1等位基因组进行了分析。患者的格雷夫斯病诊断依据为激素谱检测结果：促甲状腺激素（TSH）降低/受抑，同时游离甲状腺素（freeT4）、游离三碘甲状腺原氨酸（freeT3）水平升高，且存在促甲状腺素受体抗体（thyrotropin receptor antibodies, TRAK），辅以甲状腺超声检查结果。
研究对象于2017-2020年期间招募，主要来自布拉格内分泌研究所的门诊患者。所有患者均在该研究所接受持续随访，可为我们提供关于其疾病状态的详细长期乃至终身回顾性临床数据。
本研究采用基于因美纳（Illumina）MiSeq测序平台的二代测序技术开展单倍型分型。
针对上述3个HLA基因，我们对其第2外显子进行扩增，这是由于HLA分子的该区域决定了其抗原特性。
DNA序列扩增采用德国PentaGen Protrans公司生产的等位基因特异性预包被PCR试纸条试剂盒：PROTRANS S4 HLA-DRB1*（货号34 04）、PROTRANS S3 HLA-DQB1*（货号33 09）及PROTRANS S2 HLA-DQA1*（货号32 08），上述试剂盒均通过欧盟认证（CE 0197、CE IVD）。