Genemapper

SSR reactions were set up in a 10 μL volume of a mixture containing 25 ng of genomic DNA, 0.75 U Taq (Promega, Madison WI, USA), 1X reaction buffer, 100 μM dNTPS and 0,1 μM of each primer (S1 Table) as previously described [12 ]. SSR markers were analyzed on an ABI 3130 Genetic Analyzer. Size standard GeneScan 500 LIZ (Applied Biosystems, Foster City, CA, USA) was included with each sample to define allele sizes. Data were analyzed using GeneMapper (Applied Biosystems, Foster City, CA, USA). AFLP reactions were carried out using the AFLP Plant Mapping kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. EcoRI+A/MseI+C primer set was used for pre-selective reactions. Three primer pairs were used for the selective reactions, since they were found to be the most informative, after screening 32 primer combinations (S1 Table). PCR products were mixed with 0.1 μL molecular size standard (GeneScan 500 LIZ, Thermo Fisher Scientific, USA) and fractioned on a Genetic Analyzer ABI Prism 3130 (Applied Biosystems). Data were analyzed using GeneMapper (Applied Biosystems, Foster City, CA, USA).

The mutation screening of the PHOX2B gene (GenBank NM_003924.3) was performed as already reported (6 (link)). In particular, the three PHOX2B exons were amplified by specific primers (Supplementary Table 1) by using the GC Rich PCR System (Roche). Reaction mixes were run for 35 cycles at 95°C denaturation for 1 min, 60°C annealing for 45 s, and 72°C extension for 1 min and 30 s.
PCR fragments were purified with the SapI–ExoIII enzymatic mix by incubating at 37°C for 40′ and at 80°C for 15′ and analyzed for mutations by direct DNA sequencing using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystem) on an ABI 3100 DNA automated sequencer.
The PHOX2B 7Ala in-frame deletion (hereon 7Ala contraction) was confirmed also by using the “FAM method” (23 (link)). In detail, PCR was performed with 22F-FAM 5′-CTGACCCGGACAGCACTGGGGGCC-3′, 5′ end-labeled with FAM, and 279R 5′-GAGCCCAGCCTTGTCCAGG-3′ by the Accuprime GC kit (Life Technologies). Reaction mixes were run for 35 cycles at: 95°C denaturation for 1 min, 62°C annealing for 45 s, and 72°C extension for 45 s, followed by 20 min final extension. One microliter of the PCR product was mixed to 12 μl of formamide and 0.3 μl of ROX 500 size marker (Applied Biosystems) and loaded on the ABI 3100 DNA automated sequencer. Data were then analyzed by GeneMapper (Applied Biosystems).

HLA class II (HLA-DRB1, HLA-DQB1) typing was performed by PCR sequence-specific primer (SSP) methodology (Olerup SSP AB; Invitrogen Ltd, One Lambda Inc) following the manufacturer’s protocol.
SNP variants for IL-10 1082A>G (rs1800896), CTLA4 CT60A>G (rs3087243), TNF 308G>A (rs1800629), CD32 500 A>G (rs1801274), MAPK9 (rs4147385) were genotyped. For the CD86 gene, four biallelic SNPs were investigated: rs2715267 in the promoter region, rs2681417 in the exon 4 region, rs1129055 in the exon 7 region and rs2681401 in the untranslated transcribed region (UTR). These four biallelic SNPs were selected as candidate SNPs for the analysis, since a team from the ABIRISK consortium has previously shown that antigen presenting cells are activated when exposed to specific FVIII concentrates and that this activation correlates with CD86 expression levels [20 (link)].
All these biallelic polymorphisms were detected by PCR amplification and direct sequencing.
Regarding HMOX-1, the 5’-flanking region of the HO-1 gene containing the (GT)_n dinucleotide repeat was amplified as described elsewhere [21 (link)]. Each repeat number was calculated with GeneMapper (Applied Biosystems). To confirm the size of (GT)_n repeats, selected samples were subjected to a sequence analysis.