Online-Only Appendix Note 1

Online-Only Appendix Note 1

Online-Only Appendix Note 1

MICA-STR PCR conditions

10 l of DNA at 4 ng/l, 1.49 l PCR buffer (in 10 ml reaction buffer: 0.8 g Trizma base, 0.22 g ammonium sulphate, 0.67 ml 1M MgCl2, pH to 8.8 with HCl), 1.49 l dNTP 5 mM, 0.37 l forward primer, 0.37 l reverse primer, 0.49 l bovine serum albumen 1/10 (Sigma), 5.58 l 28% sucrose and cresol red, 0.12 l Amplitaq. The cycling conditions were: 96 C for 1 min, 94 C for 20 s, 65 C for 1 min, 72 C for 2 min, this cycle repeated 35 times.

MICA and MICB PCR conditions

10 l of DNA at 4 ng/l, 1.49 l PCR buffer (in 10 ml reaction buffer: 0.8 g Trizma base, 0.22 g ammonium sulphate, 0.67 ml 1M MgCl2, pH to 8.8 with HCl), 1.49 l dNTP 5 mM, 0.37 l forward primer, 0.37 l reverse primer, 0.49 l bovine serum albumen 1/10 (Sigma), 5.58 l 28% sucrose and cresol red, 0.12 l Amplitaq. The cycling conditions were: 95 C for 5 min, 94 C for 45 s, 65 C for 45 s (reduce by –0.5 C per cycle), 72 C for 2 min, this cycle repeated 19 times, then 94 C for 45 s, 55 C for 45 s, 72 C for 2 min, this cycle repeated 13 times, then 72 C for 10 min.

Sequencing conditions

The PCR products were then cleaned using 0.9 l Shrimp Alkaline Phosphatase (USB), 0.225 l Exonuclease I (BioLabs) and 4.9 l water per sample. Sequencing was then carried out on an Applied Biosystems (ABI) 3700. The sequencing reaction was done using 2 l PCR product, 3 l Big Dye Buffer (ABI), 1 l Big Dye Terminator (ABI), 0.25 l primer and 3.75 l water. The cycling conditions were: 96 C for 10 s, 50 C for 5 s, 60 C for 4 min, repeat this cycle 24 times, then 4 C for 5 min.

Online-Only Appendix Table 1: Combinations of MICA gene alleles and MICA-STR alleles observed in 818 families with type 1 diabetes.

All the raw data from this study is available from the Diabetes and Inflammation Laboratory

MICA-STR alleles / MICA gene alleles
MICA-STR*A4 / *001, *007, *008, *012, *018, *021, *051
MICA-STR*A5 / *008, *010, *016, *019, *027
MICA-STR*A5.1 / *002, *004, *008, *010, *018, *023
MICA-STR*A6 / *004, *006, *009, *011
MICA-STR*A9 / *002, *008, *017, *041

Online-Only Appendix Table 2: D’ values for MICA alleles, MICA microsatellite alleles and MICB alleles in relation to HLA-DRB1 alleles and HLA-DQB1 alleles. Only the MICA and MICB alleles that showed association with type 1 diabetes prior to class II conditioning are shown.

All the raw data from this study is available from the Diabetes and Inflammation Laboratory

MICA alleles / MICA-STR alleles / MICB alleles
*001 / *008 / *010 / *012 / *017 / *018 / *027 / *A5 / *A5.1 / *A6 / *A9 / *003 / *005
DRB1*01
/ 0.04 / 0.51 / 0.09 / 0.01 / 0.02 / 0.02 / 0.01 / 0.08 / 0.35 / 0.03 / 0.21 / 1.00 / 0.04
DRB1*03 / 0.61 / 0.36 / 0.01 / 0.17 / 1.00 / 0.63 / 0.21 / 0.86 / 0.42 / 0.48 / 0.82 / 0.41 / 0.32
DRB1*04 / 0.88 / 0.45 / 0.84 / 0.59 / 0.25 / 1.00 / 0.98 / 0.04 / 0.21 / 0.12 / 0.88 / 1.00 / 0.33

DRB1*06

/ 1.00 / 0.58 / 0.49 / 1.00 / 1.00 / 1.00 / 1.00 / 1.00 / 0.78 / 1.00 / 1.00 / 1.00 / 1.00
DRB1*07 / 1.00 / 0.99 / 1.00 / 1.00 / 1.00 / 1.00 / 1.00 / 0.01 / 0.53 / 0.29 / 0.06 / 0.50 / 0.45
DRB1*08 / 0.06 / 0.52 / 0.97 / 1.00 / 0.49 / 0.39 / 0.09 / 0.30 / 0.64 / 0.04 / 0.28 / 1.00 / 0.04
DRB1*09 / 1.00 / 0.63 / 0.01 / 0.43 / 0.99 / 0.97 / 0.04 / 0.13 / 0.02 / 0.62 / 0.03 / 0.05 / 0.02
DRB1*10 / 1.00 / 0.04 / 0.23 / 0.08 / 0.07 / 0.73 / 1.00 / 0.32 / 0.36 / 0.09 / 0.08 / 0.07 / 0.18
DRB1*11 / 1.00 / 0.46 / 1.00 / 1.00 / 0.06 / 0.06 / 0.08 / 0.19 / 0.35 / 0.26 / 0.29 / 1.00 / 0.24
DRB1*12 / 0.01 / 0.34 / 0.02 / 0.05 / 1.00 / 0.17 / 0.19 / 0.83 / 0.42 / 0.03 / 1.00 / 1.00 / 0.29
DRB1*13 / 1.00 / 0.47 / 0.50 / 1.00 / 1.00 / 0.11 / 0.03 / 0.17 / 0.10 / 0.06 / 0.05 / 0.12 / 0.07
DRB1*14 / 1.00 / 0.07 / 0.40 / 0.16 / 0.01 / 0.03 / 0.08 / 0.21 / 0.53 / 0.42 / 0.09 / 1.00 / 0.27
DRB1*15 / 0.01 / 0.53 / 1.00 / 0.11 / 1.00 / 0.00 / 0.02 / 0.66 / 0.42 / 0.01 / 0.99 / 1.00 / 0.56
DRB1*16 / 0.90 / 0.35 / 0.36 / 1.00 / 1.00 / 0.09 / 0.99 / 0.36 / 0.16 / 0.41 / 0.31 / 1.00 / 0.15
DRB1*0401 / 1.00 / 0.11 / 1.00 / 0.02 / 1.00 / 0.03 / 0.05 / 0.38 / 0.09 / 0.51 / 0.42 / 0.02 / 0.07
DRB1*0402 / 0.85 / 0.11 / 0.57 / 0.31 / 0.04 / 0.03 / 0.17 / 0.03 / 0.58 / 0.20 / 0.19 / 0.78 / 0.10
DRB1*0403 / 1.00 / 0.48 / 1.00 / 1.00 / 0.51 / 0.15 / 1.00 / 0.34 / 0.16 / 0.32 / 1.00 / 1.00 / 0.37
DRB1*0404 / 1.00 / 0.19 / 0.01 / 0.18 / 1.00 / 0.13 / 1.00 / 0.17 / 0.18 / 0.33 / 0.05 / 0.40 / 0.26
DRB1*0405 / 1.00 / 0.18 / 0.01 / 0.73 / 0.59 / 0.35 / 1.00 / 0.91 / 0.51 / 0.59 / 0.52 / 0.01 / 0.21
DQB1*02 / 0.87 / 0.25 / 0.87 / 1.00 / 0.86 / 0.81 / 0.40 / 0.69 / 0.22 / 0.08 / 0.80 / 0.64 / 0.02
DQB1*04 / 0.90 / 0.64 / 0.01 / 0.46 / 1.00 / 0.99 / 0.17 / 0.49 / 0.65 / 0.07 / 0.29 / 1.00 / 0.06
DQB1*05 / 1.00 / 0.99 / 1.00 / 1.00 / 1.00 / 1.00 / 1.00 / 1.00 / 0.03 / 1.00 / 0.22 / . / .
DQB1*06 / 1.00 / 1.00 / 0.18 / 0.31 / 1.00 / 1.00 / 1.00 / 0.10 / 0.02 / 0.09 / 1.00 / 1.00 / 1.00
DQB1*0301 / 0.25 / 0.18 / 0.38 / 0.09 / 1.00 / 0.17 / 0.21 / 0.02 / 0.15 / 0.28 / 0.36 / 1.00 / 0.39
DQB1*0302 / 0.92 / 0.23 / 0.64 / 0.26 / 0.07 / 0.29 / 0.56 / 0.45 / 0.20 / 0.09 / 0.12 / 0.01 / 0.21
DQB1*0303 / 0.26 / 0.33 / 0.03 / 0.10 / 0.60 / 0.76 / 0.92 / 0.38 / 0.34 / 1.00 / 0.42 / 0.54 / 0.34
DQB1*0304 / 1.00 / 0.99 / 1.00 / 1.00 / 1.00 / 1.00 / 1.00 / 1.00 / 0.98 / 1.00 / 1.00 / 1.00 / 1.00
DQB1*0305 / 1.00 / 1.00 / 1.00 / 1.00 / 1.00 / 1.00 / 1.00 / 1.00 / 1.00 / 1.00 / 0.35 / 1.00 / 1.00
DQB1*0501 / 0.86 / 0.30 / 0.24 / 0.15 / 1.00 / 0.37 / 0.19 / 0.23 / 0.32 / 0.01 / 0.22 / 1.00 / 0.05
DQB1*0502 / 1.00 / 0.12 / 1.00 / 0.02 / 0.51 / 0.02 / 0.05 / 0.45 / 0.16 / 0.21 / 0.23 / 1.00 / 0.29

DQB1*0503

/ 0.02 / 0.53 / 1.00 / 0.11 / 1.00 / 0.01 / 0.02 / 0.11 / 0.53 / 0.26 / 0.01 / 1.00 / 0.26
DQB1*0504 / 1.00 / 1.00 / 0.08 / 1.00 / 1.00 / 1.00 / 0.23 / 0.10 / 1.00 / 1.00 / 1.00 / 1.00 / 1.00
DQB1*0601 / 0.03 / 0.68 / 0.02 / 1.00 / 1.00 / 0.06 / 1.00 / 0.70 / 0.71 / 0.65 / 1.00 / 1.00 / 0.79
DQB1*0602 / 1.00 / 0.39 / 0.19 / 0.50 / 1.00 / 0.08 / 0.21 / 0.38 / 0.46 / 0.31 / 1.00 / 1.00 / 0.79
DQB1*0603 / 1.00 / 0.16 / 0.02 / 0.02 / 0.61 / 0.04 / 0.13 / 0.12 / 0.16 / 0.39 / 0.04 / 0.67 / 0.17
DQB1*0604 / 1.00 / 0.40 / 0.87 / 0.09 / 0.01 / 0.11 / 0.94 / 0.52 / 0.28 / 0.17 / 0.18 / 0.01 / 0.15

DQB1*0605

/ 1.00 / 0.99 / 1.00 / 1.00 / 1.00 / 1.00 / 1.00 / 1.00 / 1.00 / 1.00 / 0.35 / 1.00 / 1.00
DQB1*0609 / 1.00 / 0.68 / 0.81 / 1.00 / 1.00 / 0.02 / 1.00 / 1.00 / 0.69 / 0.50 / 0.38 / 1.00 / 0.07

Online-Only Appendix Table 3: r2 values for MICA alleles, MICA microsatellite alleles and MICB alleles in relation to HLA-DRB1 alleles and HLA-DQB1 alleles. Only the MICA and MICB alleles that showed association with type 1 diabetes prior to class II conditioning are shown.

MICA alleles / MICA alleles / MICA alleles
*001 / *008 / *010 / *012 / *017 / *018 / *027 / *A5 / *A5.1 / *A6 / *A9 / *003 / *005
DRB1*01 / 0.002 / 0.015 / 0.000 / 0.000 / 0.002 / 0.001 / 0.000 / 0.000 / 0.014 / 0.000 / 0.036 / 0.002 / 0.000
DRB1*03 / 0.085 / 0.067 / 0.025 / 0.002 / 0.001 / 0.007 / 0.004 / 0.038 / 0.063 / 0.013 / 0.030 / 0.001 / 0.022
DRB1*04 / 0.000 / 0.001 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000

DRB1*06

/ 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.002 / 0.000 / 0.000 / 0.001
DRB1*07 / 0.002 / 0.022 / 0.007 / 0.001 / 0.068 / 0.000 / 0.001 / 0.000 / 0.022 / 0.042 / 0.002 / 0.069 / 0.026
DRB1*08 / 0.001 / 0.008 / 0.000 / 0.000 / 0.000 / 0.000 / 0.001 / 0.000 / 0.009 / 0.000 / 0.014 / 0.000 / 0.000
DRB1*09 / 0.001 / 0.000 / 0.008 / 0.000 / 0.004 / 0.000 / 0.000 / 0.002 / 0.000 / 0.001 / 0.000 / 0.002 / 0.000
DRB1*10 / 0.000 / 0.001 / 0.000 / 0.000 / 0.001 / 0.001 / 0.002 / 0.000 / 0.000 / 0.000 / 0.000 / 0.001 / 0.000
DRB1*11 / 0.000 / 0.005 / 0.000 / 0.001 / 0.001 / 0.015 / 0.012 / 0.010 / 0.005 / 0.000 / 0.000 / 0.001 / 0.004
DRB1*12 / 0.000 / 0.002 / 0.000 / 0.000 / 0.000 / 0.005 / 0.001 / 0.001 / 0.002 / 0.000 / 0.001 / 0.000 / 0.001
DRB1*13 / 0.003 / 0.001 / 0.001 / 0.004 / 0.000 / 0.000 / 0.001 / 0.000 / 0.001 / 0.002 / 0.002 / 0.000 / 0.001
DRB1*14 / 0.000 / 0.003 / 0.001 / 0.008 / 0.000 / 0.000 / 0.000 / 0.000 / 0.003 / 0.000 / 0.001 / 0.000 / 0.001
DRB1*15 / 0.002 / 0.008 / 0.001 / 0.001 / 0.002 / 0.002 / 0.001 / 0.005 / 0.012 / 0.000 / 0.009 / 0.001 / 0.012
DRB1*16 / 0.001 / 0.000 / 0.001 / 0.000 / 0.000 / 0.001 / 0.002 / 0.000 / 0.000 / 0.000 / 0.010 / 0.000 / 0.001
DRB1*0401 / 0.008 / 0.004 / 0.120 / 0.000 / 0.000 / 0.000 / 0.000 / 0.080 / 0.002 / 0.011 / 0.006 / 0.000 / 0.003
DRB1*0402 / 0.001 / 0.003 / 0.001 / 0.000 / 0.000 / 0.016 / 0.000 / 0.000 / 0.005 / 0.004 / 0.005 / 0.000 / 0.000
DRB1*0403 / 0.000 / 0.000 / 0.000 / 0.024 / 0.000 / 0.009 / 0.000 / 0.000 / 0.000 / 0.000 / 0.001 / 0.000 / 0.003
DRB1*0404 / 0.003 / 0.002 / 0.000 / 0.001 / 0.001 / 0.000 / 0.001 / 0.000 / 0.002 / 0.001 / 0.001 / 0.000 / 0.003
DRB1*0405 / 0.001 / 0.006 / 0.002 / 0.000 / 0.000 / 0.000 / 0.000 / 0.002 / 0.005 / 0.041 / 0.001 / 0.000 / 0.000
DQB1*02 / 0.062 / 0.028 / 0.036 / 0.006 / 0.007 / 0.006 / 0.001 / 0.033 / 0.024 / 0.002 / 0.039 / 0.004 / 0.000
DQB1*05 / 0.001 / 0.009 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.001 / 0.009 / 0.001 / 0.014 / 0.000 / 0.000
DQB1*06 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.001 / . / .
DQB1*0301 / 0.000 / 0.001 / 0.000 / 0.009 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.002
DQB1*0302 / 0.000 / 0.004 / 0.002 / 0.001 / 0.003 / 0.005 / 0.004 / 0.000 / 0.003 / 0.002 / 0.002 / 0.003 / 0.034
DQB1*0303 / 0.011 / 0.019 / 0.120 / 0.000 / 0.000 / 0.005 / 0.001 / 0.085 / 0.014 / 0.001 / 0.001 / 0.000 / 0.009
DQB1*0304 / 0.000 / 0.004 / 0.000 / 0.000 / 0.231 / 0.000 / 0.000 / 0.001 / 0.004 / 0.005 / 0.044 / 0.209 / 0.002
DQB1*0305 / 0.000 / 0.001 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.001 / 0.000 / 0.008 / 0.000 / 0.002
DQB1*0501 / 0.000 / 0.001 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.001 / 0.000 / 0.001 / 0.000 / 0.002
DQB1*0502 / 0.003 / 0.010 / 0.001 / 0.000 / 0.002 / 0.000 / 0.000 / 0.001 / 0.011 / 0.000 / 0.039 / 0.002 / 0.001

DQB1*0503

/ 0.001 / 0.000 / 0.001 / 0.000 / 0.000 / 0.000 / 0.002 / 0.000 / 0.000 / 0.000 / 0.006 / 0.000 / 0.002
DQB1*0504 / 0.000 / 0.003 / 0.001 / 0.009 / 0.000 / 0.000 / 0.000 / 0.000 / 0.003 / 0.000 / 0.000 / 0.000 / 0.001
DQB1*0601 / 0.000 / 0.002 / 0.000 / 0.000 / 0.000 / 0.000 / 0.008 / 0.000 / 0.001 / 0.007 / 0.000 / 0.000 / 0.001
DQB1*0602 / 0.000 / 0.002 / 0.000 / 0.000 / 0.000 / 0.001 / 0.000 / 0.000 / 0.002 / 0.011 / 0.001 / 0.000 / 0.005
DQB1*0603 / 0.002 / 0.009 / 0.000 / 0.000 / 0.001 / 0.002 / 0.000 / 0.001 / 0.014 / 0.001 / 0.008 / 0.001 / 0.022
DQB1*0604 / 0.001 / 0.001 / 0.000 / 0.000 / 0.000 / 0.001 / 0.007 / 0.003 / 0.001 / 0.001 / 0.000 / 0.000 / 0.002

DQB1*0605

/ 0.001 / 0.005 / 0.003 / 0.004 / 0.000 / 0.000 / 0.000 / 0.001 / 0.003 / 0.000 / 0.000 / 0.000 / 0.000
DQB1*0609 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.000 / 0.001 / 0.005 / 0.001 / 0.000 / 0.002