Oligonucleotides

We selected 22 microsatellites spanning 2.44 Mbp (from HLA-E to PSMB9 gene) in the HLA region (Supplementary Table 1) harbouring the HLA class I, II, class III regions, and genotyped all patients and control subjects. Forward primers of the primer sets to amplify microsatellites were labeled by 5′ fluorescent FAM. Oligonucleotides were obtained from Greiner Bio-one. PCR and fragment analyses were performed with capillary electrophoresis using an Applied Biosystems 3730 Genetic Analyzer. Allele assignment was determined with GeneMapper Software (Thermo Fisher Scientific) and conducted as previously described [18] (link). Fragment sizes were assigned to allele names in the corresponding microsatellites. In the MICA locus, 5 MICA polymorphisms (A4, A5, A5.1, A6, A9) were determined based on the number of alanine (GCT) repeats. The A5.1 allele contained 5 GCT repeats, plus 1 extra guanine nucleotide (GCT)₂G(GCT)₃.

Coding exons of CCHCR1 were sequenced using PCR-based capillary Sanger sequencing. Oligonucleotides were purchased from Greiner Bio-One (Supplementary Table 3). PCR was performed in a reaction volume of 10 μl containing 5 ng of genomic DNA, 0.2 U of KOD FX Neo (TOYOBO Life Science, Osaka, Japan), 5 μl of 2 × PCR Buffer, 2 μl of dNTP (2 mM each), and 0.2 μM (final concentration) of each of the primers. The thermal cycling profile was as follows: initial denaturation at 94 °C for 2 min and 35 rounds of amplification at 98 °C for 10 s, then 59–65 °C (depending on primer set; see Supplementary Table 3) for 30 s and 68 °C for 1 min. PCR products were purified using an AMPure XP (Beckman Coulter, Fullerton, CA, USA), according to the manufacturer's protocol. Purification and sequencing of the PCR products were carried out using a BigDye Terminator v3.1 Cycle Sequencing kit (Thermo Fisher Scientific) and BigDye XTerminator Purification Kit (Thermo Fisher Scientific), following the manufacturer's instructions. Automated electrophoresis was performed with an ABI PRISM 3730 Genetic Analyzer (Thermo Fisher Scientific). Sequencing data were analyzed using Sequencher (v. 5.1, Gene Codes Corporation, Ann Arbor, MI, USA). Genomic coordinates for all variants were called using the UCSC Genome Browser assembly GRCh37/hg19 (http://genome.ucsc.edu/).