Introduction
Methodology
Human ear phenotypes and study cohort
Selection of targeted DNA variants
SNP genotyping assay
Assay position | SNP ID | Chr number and region | Gene | Major allele | Minor allele | PCR primer sequences | Primer conc. (µM) | Product size | SBE primer sequence, length of SBE primer, and direction | Detected allele | SBE primer conc. (µM) |
---|---|---|---|---|---|---|---|---|---|---|---|
Plex1_1 | rs10212419 | 3, intronic | MRPS22 | CC | TT | F-CTTTGGGCTCAACCCGACTA R-TATGTGGAATGGGCTCTCCC | 0.3 | 235 | TTTTTTTTTTGCACACGTAGTATCTTGTATAACC, 34, R | T/C | 0.2 |
Plex1_2 | rs17023457 | 1, intronic | TBX15 | TT | CC | F-TGGAGACTCTGAGACAACCTGA R-CCCACTCCTCACCAGAAACT | 0.3 | 296 | TTTTTTTTTTTTTGATACCGACCACTAACTAATCAACA, 38, F | T/C | 0.2 |
Plex1 _3 | rs13397666 | 2, intronic | EDAR | AA | GG | F-CAGGTCTGAACCGTAGCCAG R-CAGAGATGGCCTGAACCTCC | 0.3 | 188 | TTTTTTTTTTTTTTTTTTTTATAGGTCGGCGAGGTTCC, 38, F | A/G | 0.2 |
Plex1 _4 | rs7567615 | 2, intronic | SH3RF3 | GG | AA | F-CTGTGAGGTCAACTGAGCGG R-CCCACAATGACAGCCACCTT | 0.3 | 164 | TTTTTTTTTTTTTTTTTTTTCCAACGATCAGAAAATAAACCC, 42, R | A/G | 0.2 |
Plex1 _5 | rs3827760 | 2, missense | EDAR | TT | F-AGAGTTGCATGCCGTCTGTC R-CCACGGAGCTGCCATTTGAT | 0.3 | 159 | TTTTTTTTTTTTTTTTTTTCACGTACAACTCTGAGAAGGCTG, 38, R | T/C | 0.2 | |
Plex1 _6 | rs7428 | 2, 3′UTR | TGOLN2 | TT | CC | F-TCAAACATGAAGTCTGGTGCATT R-ACCCCTGTTAGGAAGGTTGG | 0.3 | 253 | TTTTTTTTTTTTTTTTTTTTTTGCTTACTGGCAGTTTGACATACTA, 46, F | T/C | 0.2 |
Plex1 _7 | rs868157 | 1, intergenic | TF binding site LOC107985447 | TT | GG | F-AGCCCTTGAATGAGGGTTGG R-GGGGGCTTGCACATCATAGA | 0.3 | 229 | TTTTTTTTTTTTTTTTTTTTTTATTATCTACCATACCAAAACTATGAGCT, 50, F | G/T | 0.2 |
Plex1 _8 | rs2080401 | 2, intronic | SP5 | AA | CC | F-TAGTAGAGTAGCCCACAGA R-CTGGTCTTGAACTCCTGA | 0.3 | 122 | TTTTTTTTTTTTTTTTTTTTTTTTTGCAACTAGTAGAGTAGCCCACAGAA, 50, F | A/C | 0.2 |
Plex1 _9 | rs7873690 | 9, regulatory region | SLC4A1PP1 | CC | TT | F-TTCCGTTGAAGGGTGCTGTA R-CCCTGAAACTGGAACAGAGCC | 0.3 | 300 | TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCAGGGGAATCCCAGGAG, 54, F | T/C | 0.2 |
Plex 1_10 | rs1960918 | 4, intronic | LRBA | TT | CC | F-AACAAGAAACCAAGAACCCAAATA R-TCCTTCTTCCTGTCTGTCCTCTTA | 0.3 | 254 | TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAGATAATTGAGTGAATCTCGGTAA, 58, F | T/C | 0.2 |
Plex2_1 | rs3818285 | 10, intronic | XPNPEP1 | AA | GG | F-GAACAGAGTCACAACTGGGCTA R-ACCTTATTGACTCGGGTGCT | 0.4 | 215 | TTTTTTTTTTTTTTAAGGTGGACAGCTGAGCTCC, 34, R | G/A | 0.4 |
Plex2_2 | rs6845263 | 7 regulatory region | FLJ20021 | CC | TT | F-GCACCTCATCACTCTCTGCC R-AGGTTAGAAAAACTAACCCAGACT | 0.4 | 285 | TTTTTTTTTTCATCTGTATGTGTGCTGTGTTTGA, 34, F | T/C | 0.4 |
Plex2 _3 | rs2378113 | 2, 5′ UTR | GCC2 | AA | GG | F-TTTTAGTGTGCGCAATCGCC R-AGCCCACAGATCAGAATCCC | 0.4 | 208 | TTTTTTTTATAAAGCAGTCTAAGAAGGTTTATATAGTG, 38, F | G/A | 0.4 |
Plex2_4 | rs10923574 | 1, regulatory region | WDR3 | AA | CC | F-ACCCTATGAAAAGAGCATGTAGT R-AATCACGTAGACTGAGGGGA | 0.4 | 263 | TTTTTTTTTTTTTTTAACAGCCTTTTCAAGAAATACCTATTA, 42, F | A/C | 0.4 |
Plex2 _5 | rs1619249 | 2, intergenic | LOC100287225 | TT | CC | F-CTTGATCTCCTGACCTCTT R-GTGGACTTTACATTTACTCTGA | 0.4 | 84 | TTTTTTTTTTTTTTTTTTTTTGTGGGCGGATAGGAGGC, 38, R | T/C | 0.4 |
Plex2 _6 | rs9866054 | 3, intronic | MRPS22, FOXL2 | GG | AA | F-TTGAGGGCTTCTCTTGTGGC R-CCCACTGTCTTAAAGTAGCCCATT | 0.4 | 109 | TTTTTTTTTTTTTTTTTTTTTTTTAGCTGTTTTCTAGGCTGGATTG, 46, F | G/A | 0.2 |
Plex2 _7 | rs263156 | 6, intronic | GPR126, LOC153910 | CC | AA | F-CAAAGGCCCATGCAGCTACT R-TTGGAAGGCACATCAACCAC | 0.4 | 181 | TTTTTTTTTTTTTTTTTTTTTTTTTTTCTCATCTACCCTATCATTCCACC, 50, R | A/C | 0.2 |
Plex2_8 | rs260674 | 2, intronic | EDAR | AA | GG | F-ACTCAAAACCGAGTGTCCCG R-TGAACCCCGCCAATGTCCTA | 0.4 | 121 | TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTTTGGTTACGTCTGCCC, 54, F | G/A | 0.1 |
Plex2 _9 | rs10192049 | 2, intronic | Antisense to MYO3b | AA | GG | F-TCGTGGCAAGTTACGTGTGTA R-AATGCTTGGTGCACGGTAGG | 0.4 | 281 | TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGTATCAGTCCATATGCCTTCACA, 58, R | G/A | 0.4 |
Plex2_10 | rs13427222 | 2, intronic | EDAR | AA | GG | F-GGCCTGATGGTTCGGAGTTA R-AAGGAGAG TAGCGCTGGGT | 0.4 | 277 | TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCCAGCACCTTGCCTCCC, 62, R | A/G | 0.4 |
Plex2_11 | rs1879495 | 11, intergenic | SULT1C2P1 | CC | AA | F-AAGTGACCTCCTGGACTTGG R-GCACCAGCAGGGGAAAGTA | 0.4 | 299 | TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGGTAGAACTGGAACAAAATCTT, 66, R | A/C | 0.4 |
Capillary electrophoresis and allele calling
Statistical analysis
Population analysis
Association testing
Prediction modelling
Results
Population data
SNaPshot™ multiplex SNP genotyping assay and screening of genotypes
SNP associations testing in Punjab population
B | Std. error | p-value | Odds ratio | |||
---|---|---|---|---|---|---|
Lobe size | Small | −7.665 | 1.8989 | .000 | .100 | |
Medium | −4.471 | 1.8518 | .016 | .214 | ||
Large | References | |||||
rs17023457 | TT | 1.115 | .5556 | .045 | 3.049 | |
CT | 1.087 | .6193 | .079 | 2.965 | ||
CC | References | |||||
rs13397666 | AA | −.477 | .4120 | .247 | .621 | |
AG | −.789 | .3942 | .043 | .454 | ||
GG | References | |||||
rs1960918 | TT | −.387 | .4297 | .368 | .679 | |
CT | −.765 | .3758 | .042 | .466 | ||
CC | References | |||||
rs1619249 | TT | −1.074 | .7516 | .153 | .342 | |
CT | −1.716 | .7965 | .031 | .180 | ||
CC | References | |||||
rs9866054 | GG | −.790 | .4227 | .062 | .454 | |
AG | −.979 | .5011 | .041 | .376 | ||
AA | References | |||||
rs13427222 | AA | −1.509 | .5779 | .009 | .221 | |
AG | −1.900 | .5955 | .001 | .150 | ||
GG | References | |||||
rs1878495 | CC | −.784 | .4071 | .044 | .457 | |
AC | −.274 | .4000 | .493 | .760 | ||
AA | References | |||||
Lobe attachment | Presence | −.339 | 1.9358 | .861 | .712 | |
Intermediate | 2.334 | 1.9441 | .049 | 5.318 | ||
Free | References | |||||
rs7873690 | CC | .649 | .4071 | .111 | 1.914 | |
CT | .976 | .4860 | .045 | 2.654 | ||
TT | References | |||||
rs1960918 | TT | −.470 | .4226 | .266 | .625 | |
CT | −.706 | .3789 | .042 | .493 | ||
CC | References | |||||
rs1619249 | TT | 3.194 | .9180 | .001 | 4.376 | |
CT | 2.961 | .9452 | .002 | 1.919 | ||
CC | References | |||||
rs13427222 | AA | −1.400 | .6092 | .022 | .246 | |
AG | −1.390 | .6148 | .024 | .249 | ||
GG | References | |||||
Anti-tragus size | Absent | .535 | 1.7502 | .760 | 1.708 | |
Average | 2.808 | 1.7572 | .049 | 7.579 | ||
Prominent | References | |||||
rs868157 | TT | 1.641 | .8342 | .049 | 5.159 | |
GT | 1.086 | .8747 | .214 | 2.963 | ||
GG | References | |||||
rs7873690 | CC | −.841 | .3868 | .030 | .431 | |
CT | −1.115 | .4580 | .015 | .328 | ||
TT | References | |||||
rs13427222 | AA | 1.017 | .5084 | .045 | 2.765 | |
AG | .612 | .5176 | .237 | 1.844 | ||
GG | References | |||||
Tragus size | Absent | .932 | 1.8039 | .605 | 2.540 | |
Average | 4.112 | 1.8261 | .024 | 6.083 | ||
Prominent | References | |||||
rs17023457 | TT | 1.155 | .5747 | .044 | 3.175 | |
CT | 1.148 | .6366 | .071 | 3.151 | ||
CC | References | |||||
rs868157 | TT | 1.655 | .8096 | .041 | 5.235 | |
GT | 1.312 | .8559 | .125 | 3.714 | ||
GG | References | |||||
rs7428 | TT | −.839 | .3622 | .021 | .432 | |
CT | −.683 | .3191 | .032 | .505 | ||
CC | References | |||||
rs7873690 | CC | .579 | .3989 | .147 | 1.784 | |
CT | .897 | .4783 | .041 | 2.452 | ||
TT | References | |||||
rs684523 | CC | .533 | .3095 | .085 | 1.704 | |
CT | .653 | .3153 | .038 | 1.922 | ||
TT | References | |||||
rs1619249 | TT | 1.724 | .7839 | .028 | 5.609 | |
CT | 1.369 | .8121 | .092 | 3.931 | ||
CC | References | |||||
rs263156 | CC | .398 | .3492 | .254 | 1.489 | |
AC | −.683 | .3464 | .049 | .505 | ||
AA | References | |||||
Superior helix rolling | Under folded | −.402 | 1.8691 | .830 | .669 | |
Partial folded | 3.424 | 1.8775 | .048 | 7.691 | ||
Over folded | References | |||||
rs13397666 | AA | .221 | .4244 | .603 | 1.247 | |
AG | .806 | .4134 | .041 | 2.240 | ||
GG | References | |||||
rs7567615 | GG | .898 | .3844 | .020 | 2.454 | |
AG | .283 | .4915 | .565 | 1.327 | ||
AA | References | |||||
Posterior helix rolling | Under folded | .326 | 1.7224 | .850 | 1.386 | |
Partial folded | 2.981 | 1.7317 | .045 | 5.716 | ||
Over folded | References | |||||
rs7428 | TT | −.839 | .3622 | .021 | .432 | |
CT | −.683 | .3191 | .032 | .505 | ||
CC | References | |||||
rs684523 | CC | .533 | .3095 | .085 | 1.704 | |
CT | .653 | .3153 | .038 | 1.922 | ||
TT | References | |||||
rs1619249 | TT | 1.724 | .7839 | .028 | 5.609 | |
CT | 1.369 | .8121 | .092 | 3.931 | ||
CC | References | |||||
rs263156 | CC | .398 | .3492 | .254 | 1.489 | |
AC | −.683 | .3464 | .049 | .505 | ||
AA | References | |||||
Antihelix folding | Under folded | −.699 | 1.8591 | .707 | .497 | |
Partial folded | 2.986 | 1.8709 | .031 | 4.806 | ||
Over folded | References | |||||
rs2080401 | AA | .474 | .4031 | .239 | 1.607 | |
AC | .915 | .3790 | .016 | 2.496 | ||
CC | References | |||||
rs260674 | AA | −1.663 | .9087 | .047 | .190 | |
AG | −1.511 | .9323 | .105 | .221 | ||
GG | References | |||||
Antihelix superior crus | Flat | −6.903 | 1.9609 | .000 | .001 | |
Intermediate | −4.480 | 1.9352 | .021 | .011 | ||
Extended | References | |||||
rs17023457 | TT | −1.048 | .6025 | .082 | .351 | |
CT | −1.462 | .6622 | .027 | .232 | ||
CC | References | |||||
rs7567615 | GG | .780 | .3618 | .031 | 2.182 | |
AG | .635 | .4815 | .187 | 1.887 | ||
AA | References | |||||
rs1960918 | TT | 1.230 | .4330 | .005 | 3.420 | |
CT | .842 | .3729 | .024 | 2.321 | ||
CC | References | |||||
rs9866054 | GG | −.933 | .4727 | .048 | .393 | |
AG | −.790 | .5549 | .154 | .454 | ||
AA | References | |||||
rs10192049 | AA | −.939 | .4278 | .028 | .391 | |
AG | −.503 | .4265 | .238 | .605 | ||
GG | References | |||||
rs13427222 | AA | −1.322 | .6336 | .037 | .267 | |
AG | −1.348 | .6419 | .036 | .260 | ||
GG | References | |||||
rs1878495 | CC | −.811 | .4275 | .048 | .444 | |
AC | .014 | .4231 | .974 | 1.014 | ||
AA | References | |||||
Darwin tubercle | Absent | 2.221 | 28.9650 | .049 | 6.000 | |
Degree of Tubercle | 2.587 | 28.9650 | .999 | 1.076 | ||
Prominent | References | |||||
rs13397666 | AA | 1.107 | .7257 | .127 | 3.026 | |
AG | 1.244 | .6863 | .049 | 3.471 | ||
GG | References | |||||
rs260674 | AA | −2.081 | .9516 | .029 | .125 | |
AG | −1.624 | .9752 | .096 | .197 | ||
GG | References | |||||
AA | 0b | 1 | ||||
Crus helix expression | Less | −.171 | 1.6985 | .920 | .843 | |
Prominent | 1.857 | 1.7030 | .048 | 6.402 | ||
Extended | References | |||||
rs7428 | TT | −.695 | .3582 | .052 | .499 | |
CT | −.639 | .3055 | .036 | .528 | ||
CC | References | |||||
rs2080401 | AA | −.672 | .3812 | .048 | .510 | |
AC | −.352 | .3529 | .319 | .703 | ||
CC | References | |||||
rs7873690 | CC | −.741 | .4060 | .048 | .476 | |
CT | −.802 | .4749 | .091 | .449 | ||
TT | References | |||||
Ear protrusion | Under folded | −1.488 | 1.7271 | .044 | .226 | |
Partial folded | .861 | 1.7263 | .618 | 2.366 | ||
Over folded | References | |||||
rs263156 | CC | −.639 | .3487 | .047 | .528 | |
AC | −.273 | .3401 | .422 | .761 | ||
AA | References | |||||
rs1878495 | CC | −.746 | .3985 | .041 | .474 | |
AC | −.596 | .3887 | .125 | .551 | ||
AA | References |
Prediction modelling accuracy
Model | Phenotype | Sensitivity% | Specificity% | PPV% | NPV% |
---|---|---|---|---|---|
Multinomial | Small lobe size | 56.8 | 81.5 | 62.3 | 77.8 |
Medium lobe size | 76.9 | 50.3 | 63 | 66.3 | |
Large lobe size | 18.1 | 99.1 | 56.7 | 90.4 | |
Attached ear lobe | 24.1 | 99.1 | 56.7 | 90.4 | |
Average attachment | 56.8 | 81.5 | 62.3 | 77.8 | |
Free ear lobe | 76.9 | 50.3 | 63 | 66.3 | |
Absent antitragus | 78.2 | 51.6 | 64.3 | 67.6 | |
Average antitragus | 19.4 | 95.8 | 58 | 91.7 | |
Large antitragus | 58.1 | 82.8 | 63.6 | 79.1 | |
Absent tragus | 49.2 | 73.95 | 59 | 76.95 | |
Average tragus | 37 | 89.65 | 56.8 | 83.7 | |
Large tragus | 65.7 | 64.85 | 61.3 | 71.1 | |
Under folded posterior helix | 76.25 | 50.2 | 62.8 | 65.55 | |
Partial folded posterior helix rolling | 18.3 | 96.8 | 54.65 | 90.65 | |
Over folded posterior helix rolling | 56.3 | 81.1 | 61.6 | 77.5 | |
Underfolded superior helix rolling | 61.75 | 61.375 | 60.15 | 70.225 | |
Partial folded superior helix rolling | 27.1 | 93.725 | 54.05 | 86.65 | |
Over folded superior helix rolling | 60.1 | 72.125 | 60.45 | 73.5 | |
Underfolded antihelix folding | 68.025 | 55.0875 | 60.725 | 66.8625 | |
Partially folded antihelix folding | 22.15 | 95.7625 | 52.675 | 88.125 | |
Over folded antihelix folding | 57.3 | 75.7625 | 60.025 | 74.7 | |
Flat antihelix superior crus | 53.38 | 69.76 | 59.38 | 74.71 | |
Average antihelix superior crus | 33.7 | 91.01 | 55.88 | 84.68 | |
Extended antihelix superior crus | 63.83 | 67.28 | 61.02 | 71.9 | |
Absent Darwin tubercle | 61.75 | 61.38 | 60.15 | 70.23 | |
Average Darwin tubercle | 27.1 | 93.73 | 54.05 | 86.65 | |
Prominent Darwin tubercle | 60.1 | 72.13 | 60.45 | 73.5 | |
Small Crus helix expression | 60.56 | 65.92 | 61.36 | 73.96 | |
Medium crus helix expression | 30.68 | 91.71 | 58.09 | 86.96 | |
Large crus helix expression | 63.3 | 72.01 | 62.66 | 74.5 | |
Flat ear | 48.82 | 67.96 | 60.3 | 75.43 | |
Medium protrusion | 31.02 | 90.11 | 58.02 | 85.19 | |
Large protrusion | 64.51 | 66.97 | 62.33 | 72.78 |