Sequencher version 4

Genotyping was performed with the OmniExp-12, v1.0 DNA Analysis BeadChip (Illumina Inc., San Diego, CA) according to the manufacturer’s instructions. SNP array data were subjected to homozygosity mapping with the Homozygosity Mapper software using only homozygous stretches of 15 alleles or longer [7 (link)]. Whole exome sequencing was accomplished according to the Nimblegen (Nimblegen v2.0, Roche Nimblegen, Indianapolis, IN) and the Extended Nextera Rapid-Capture Exome kit (Illumina, San Diego, CA, USA) protocols on an Illumina HiSeq 2000. Paired end sequence reads were aligned against the reference human genome (UCSC hg19). Exome data analysis was accomplished as previously described [8a (link), 8b (link)]. Variants were visually inspected with the Integrative Genomics Viewer (IGV) [9 (link)]. Mutation confirmation was done with Sanger sequencing using an ABI BigDye Terminator Cycle Sequencing Kit on an ABI 3730 sequencer. Sequence traces were analyzed via Sequencher (version 4.2; Gene Codes Corporation, Ann Arbor, MI, USA).
Nucleotide and protein positions are based on the following accession numbers from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) SLC25A46, NM_138773 and NP_620128. Variant positions within the cDNA are numbered using the A of the translation initiation codon as position 1.

Nucleotide sequence variation of the HLA-G 3’UTR was evaluated by direct sequencing of a 343-bp fragment encompassing the genomic positions +2885 through to +3228, using PCR primers described by (Sizzano et al., 2012 (link)). Briefly, the 3’UTR region was amplified using HLAG_3UTRF (5’-TCACCCCTCACTGTGACTGA-3’) and HLAG_3UTRR (5’-CCCATCAA-TCTCTCTTGGAAA-3’) primers with the following thermocycling conditions: 95°C for 15 min followed by 30 cycles of 93°C for 1 min, 58°C for 1 min, 72°C for 1 min. A final extension step was carried out at 72°C for 10 min. Purified amplicons using the Invitek MSB Spin PCRapace cleanup kit (Berlin, Germany) were sequenced in both directions by capillary electrophoresis using an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, California, USA) using the same PCR primers. The chromatograms obtained were analysed using Sequencher version 4.10.1 (Gene Codes Corporation, Ann Arbor, Michigan, USA) and sequences were aligned with an available HLA-G 3’UTR reference sequence (GenBank Accession number NG_029039.1) to identify known polymorphic positions (Castelli et al., 2010 (link)) and any other polymorphism that had not been previously described.