Table 2. Cytokine promoter allele frequencies in donor–recipient pairs.
| TNF (n = 152) | LTA (n = 152) | TGFB1 (n = 148)a | |||
|---|---|---|---|---|---|
| Alleleb | Number (%) | Alleleb | Number (%) | Alleleb | Number (%) |
| p001 | 100 (65.8) | p001 | 71 (46.7) | p001 | 48 (32.4) |
| p002 | 18 (11.9) | p00201 | 16 (10.5) | p002 | 5 (3.4) |
| p003 | 5 (3.3) | p00202 | 0 (0) | p003 | 79 (53.4) |
| p004 | 1 (0.7) | p003 | 8 (5.3) | p004 | 0 (0) |
| p005 | 2 (1.3) | p004 | 13 (8.6) | p005 | 0 (0) |
| p006 | 16 (10.5) | p005 | 4 (2.6) | p006 | 7 (4.7) |
| p007 | 0 (0) | p006 | 2 (1.3) | p007 | 0 (0) |
| p008 | 6 (3.9) | p007 | 18 (11.8) | p008 | 0 (0) |
| p009 | 4 (2.6) | p008 | 0 (0) | p009 | 0 (0) |
| p009 | 0 (0) | p010 | 0 (0) | ||
| p010 | 0 (0) | p011 | 0 (0) | ||
| p011 | 12 (7.9) | p012 | 0 (0) | ||
| p012 | 0 (0) | p013 | 0 (0) | ||
| p013 | 0 (0) | p014 | 7 (4.7) | ||
| p014 | 0 (0) | p015 | 0 (0) | ||
| p015 | 0 (0) | p016 | 1 (0.7) | ||
| p016 | 3 (2.0) | p017 | 1 (0.7) | ||
| p017 | 2 (1.3) | ||||
| p018 | 2 (1.3) | ||||
| p019 | 1 (0.7) | ||||
SNP, single nucleotide polymorphism; TNF, tumor necrosis factor.
There was insufficient sample to determine the TGFB1 promoter allele sequence from one of the donor–recipient pairs.
Promoter allele sequences and nomenclature are described in the Tables S1–S3, Supporting information. The frequency of alleles for each of these cytokines is not significantly different in the patients and in the donors (data not shown). The TNF regulatory region (−1075 to +161), LTA regulatory region and gene (−2434 to +743) and TGFB1 regulatory region and exon 1 (−1825 to +1252) were amplified from genomic DNA as described (14, 21). Amplified TNF DNA was purified using AMPure (Agencourt, Beverly, MA), and amplified LTA and TGFB1 DNA were purified using Microcon Ultracel YM-100(Millipore, Bedford, MA) as per the manufacturer's protocol. Purified TNF and LTA amplification products were sequenced with the Big Dye Terminator sequencing kit (PE Applied Biosystems, Foster City, CA) as described (14) with the following modifications: reaction volume was increased (15 μl) and included Big Dye Reagent (1 μl) and Better Buffer (5 μl) (The Ge Company, San Francisco, CA). Purified TGFB1 amplification products were sequenced as described (21). Reactions were performed in an MJ Research PTC-225 thermocycler (BioRad, Hercules, CA, USA) as per the PE Applied Biosystems protocol. Reactions were run on an ABI3730 automated DNA sequencer (PE Applied Biosystems) according to the manufacturer's protocol. Data were analyzed with Sequencher 5.1 software (Gene Codes Corporation, Ann Arbor, MI) as described (14, 21). LTA alleles were established by amplification and sequencing of restriction enzyme-digested genomic DNA. This used the restriction fragment length polymorphisms (RFLP) created by the SNPsat +253G/A (NcoI RFLP; dbSNP rs#909253) and/or at +369G/C (BskHiKA1 RFLP; dbSNP rs#746868). Genomic DNA (1 μg) was digested in a reaction (40 μl) containing buffer (1 ×), bovine serum albumin (0.4 μl) and the appropriate restriction enzyme (4 μl) as per the manufacturer's protocol (New England Biolabs, Beverly, MA) with the exceptions that the NcoI digest was incubated for 5 h and the BskHiKA1 digest was incubated for 18 h. Digested genomic DNA was purified using the GeneClean Turbo Kit (MP Biomedicals, Solon, OH) as per the manufacturer's protocol with the exception that the DNA was eluted in water (80 μl). Isolated LTA alleles were amplified and sequenced from purified digested genomic DNA as described above. LTA alleles for which RFLP was not possible and TNF alleles, where necessary, were isolated by cloning. Amplified products were cloned into the TA cloning vector (Invitrogen, Carlsbad, CA) as per the manufacturer's protocol. Amplified products were sequenced from multiple clones as described above. Allelic genotypes reported for TGFB1 were presumed to be a combination of previously published promoter alleles (21). TGFB1 allelic genotypes that could not be explained as a combination of known alleles were found to be heterozygous for a 3-bp insertion at position −1550 (dbSNP rs#11466313). To establish novel allelic sequences, an allele-specific polymerase chain reaction was designed to amplify alleles with the 3-bp (AGG) insertion at position −1550. Briefly, reactions (100 μl) contained genomic DNA (300 ng), primers −1550AGG (5′-AGGGCAGGGACATGAGGAGG-3′) and 3.2 (5′-TTCTTCTGCCAGTCACTTCCT-3′) (30 pm each), Platinum Taq HiFi (7.5 U) (Invitrogen), MgSO4 (2 mM), dNTPs (0.2 mM each), dimethyl sulfoxide (5%) and 10× Taq HiFi buffer (1 ×). Reactions were performed in an MJ Research PTC-225 thermocycler (BioRad) by denaturation (95°C, 10 min, 1 cycle) and amplification (95°C, 30 s; 56°C, 30 s; 68°C, 3 min; 35 cycles), followed by a final extension (68°C, 20 min, 1 cycle). Amplified DNA product was purified and sequenced as described above. In some instances, allelic sequences obtained using this method did not establish a complete allele type as some genotypes also exhibit a dimorphism (−1570A/G; dbSNP rs#2803457) upstream of the 3-bp insertion at position −1550. For these genotypes, the region from −1825 to +10 was amplified as described (21). Amplified DNA was cloned using the TOPO-TA cloning vector (Invitrogen) as per the manufacturer's protocol. Cloned DNA was sequenced as described above and combined with the data generated from the allele-specific amplification to establish the complete promoter allele sequence.