Methods for early identification of bone healing ability in injured patients
The present invention relates to the discovery that the expression levels of some RNA molecules, comprising messenger RNA (mRNA), non-coding RNA (ncRNA) and/or microRNA (miRNA), and protein can be used as a diagnostic signature to predict or monitor the bone healing ability in an acutely injured subject or in a chronic nonunion subject. In certain embodiments, the invention relates to methods and compositions useful for differentiating between a nonunion, slow healing, and/or normal healing of a fractured bone and treatment recommendations. The invention further includes a kit comprising biomarker probes for assessing the bone healing ability in an acutely injured subject or in a nonunion subject after receiving therapeutic treatment.
1 . A method of promoting bone healing in a subject with a fractured bone, the method comprising:
providing a nonunion-mitigating intervention to the subject when a difference in the level, or a difference in change of the level, of at least one RNA biomarker in a subject's sample as compared to the level of the at least one RNA biomarker in a reference sample is indicative of a nonunion or slow healing of the fractured bone in the subject, wherein the difference in level, or the difference in change of level, is determined by:
(i) comparing the level of the at least one RNA biomarker in the sample from the subject to a baseline level of the at least one RNA biomarker in the reference sample, wherein a difference in the level of the at least one RNA biomarker in the subject's sample as compared to the level in the reference sample is indicative of a nonunion or slow healing of the fractured bone in the subject; or
(ii) comparing the change of the level of the at least one RNA biomarker between samples collected from the subject at two or more different times to a baseline change of the level of the at least one RNA biomarker in reference samples collected at different times, wherein a difference in the amount of change of the at least one RNA biomarker in the subject's samples as compared to the reference samples is indicative of a nonunion or slow healing of the fractured bone in the subject;
wherein the at least one RNA biomarker is selected from the group consisting of SEQ ID NOs: 1-1684 and 1830-1838.
2 . The method of claim 1 , wherein the difference in the level, or the difference in change of the level, of the at least one RNA biomarker is determined by reverse transcription, PCR, microarray, next generation sequencing, nuclease protection, probe hybridization to one or more molecular copies of the at least one RNA biomarker, pyrosequencing, or primer extension.
3 . The method of claim 1 , wherein the level, or change in level, of the at least one RNA biomarker is at least 1.1-fold higher or lower than the level, or change in level, of the at least one RNA biomarker in the reference.
4 . The method of claim 1 , wherein the bone healing does not occur or initiate about 9 months or less after injury or after a nonunion-mitigating intervention.
5 . The method of claim 1 , wherein the bone healing does not occur or initiate about 4 months or less after injury or after a nonunion-mitigating intervention.
6 . The method of claim 1 , wherein the bone healing does not occur or initiate about 2 weeks after injury or after a nonunion-mitigating intervention.
7 . The method of claim 1 , wherein the bone healing ability is assessed at one or more time periods.
8 . The method of claim 7 , wherein the assessment is done during or before the fourth month of healing.
9 . The method of claim 1 , wherein the nonunion-mitigating intervention comprises an invasive surgical treatment comprising bone graft, removal of scar tissue, and/or immobilization of the fracture with metal plates, rods and/or pins.
10 . The method of claim 1 , wherein the nonunion-mitigating intervention comprises a non-invasive treatment comprising electrical stimulation, ultrasound, and/or immobilization of the fracture with specialized braces.
11 . The method of claim 1 , wherein the at least one RNA biomarker is at least one selected from the group consisting of a messenger RNA, a non-coding RNA, and a microRNA.
12 . The method of claim 1 , wherein the at least one RNA biomarker selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 30, 31, 53, 64, 75, 78, 89, 104, 106, 109, 126, 132, 134, 189, 191, 194, 228, 233, 239, 270, 279, 283, 290, 291, 293, 299, 344, 347, 349, 371, 388, 400, 401, 408, 413, 442, 449, 453, 459, 471, 497, 512, 513, 515, 518, 519, 520, 521, 522, 523, 525, 527, 528, 529, 531, 533, 539, 540, 541, 543, 548, 549, 550, 551, 552, 554, 556, 618, 654, 677, 702, 1520, 1549 and 1838 indicates nonunion, slow healing or normal healing after acute injury, and/or indicates successful, no or slow healing after a nonunion-mitigating intervention.
13 . The method of claim 12 , wherein the level of the at least one RNA biomarker being at least 1.1-fold higher or lower than the level of the RNA biomarker in the reference indicates nonunion, slow healing or normal healing after acute injury, and/or indicates successful, no or slow healing after a nonunion-mitigating intervention.
14 . The method of claim 1 , wherein the at least one RNA biomarker selected from the group consisting of SEQ ID NOs: 9, 24, 45, 103, 124, 164, 219, 314, 351, 516, 517, 526, 534, 535, 542, 553, 562, 563, 572, 573, 582, 598, 599, 602, 609, 619, 620, 624, 625, 634, 652, 653, 655, 656, 676, 682, 698, 700, 701, 703, 721, 722, 723, 724, 725, 729, 753, 759, 760, 762, 764, 765, 766, 768, 769, 770, 771, 777, 781, 785, 789, 791, 794, 806, 814, 816, 824, 844, 850, 855, 862, 864, 867, 885, 889, 910, 911, 912, 914, 915, 917, 918, 931, 934, 938, 954, 958, 961, 962, 966, 968, 985, 996, 1004, 1024, 1027, 1030, 1032, 1035, 1051, 1054, 1063, 1066, 1068, 1069, 1070, 1071, 1073, 1074, 1075, 1080, 1086, 1087, 1096, 1100, 1109, 1110, 1111, 1114, 1115, 1126, 1141, 1151, 1153, 1154, 1180, 1201, 1216, 1228, 1244, 1245, 1277, 1279, 1281, 1283, 1286, 1288, 1293, 1318, 1337, 1363, 1382, 1405, 1406, 1431, 1437, 1489, 1493, 1499, 1500, 1501, 1502, 1506, 1508, 1512, 1513, 1514, 1515, 1516, 1517, 1522, 1523, 1524, 1526, 1527, 1528, 1529, 1531, 1532, 1533, 1534, 1535, 1536, 1539, 1541, 1542, 1543, 1544, 1546, 1547, 1548, 1551, 1552, 1554, 1555, 1561, 1562, 1565, 1568, 1575, 1576, 1579, 1582, 1584, 1585, 1597, 1598, 1599, 1600, 1601, 1605, 1607, 1608, 1609, 1610, 1626, 1627, 1628, 1637, 1640, 1643, 1645, 1648, 1649, 1652, 1654, 1656, 1657, 1662, 1665, 1677, 1830, 1831, 1832, 1833, 1834, 1835, 1836, and 1837 indicates nonunion, slow healing or normal healing after acute injury, and/or indicates successful, no or slow healing after a nonunion-mitigating intervention.
15 . The method of claim 14 , wherein the level of the at least one RNA biomarker being at least 1.1-fold higher or lower than the level of the RNA biomarker in the reference indicates nonunion, slow healing or normal healing after acute injury, and/or indicates successful, no or slow healing after a nonunion-mitigating intervention.
16 . The method of claim 1 , wherein the differential expression of at least one RNA biomarker selected from the group consisting of SEQ ID NOs: 1, 2, 4, 5, 6, 7, 9, 24, 30, 45, 53, 64, 75, 78, 89, 99, 103, 104, 109, 124, 126, 132, 134, 164, 191, 194, 219, 228, 239, 270, 279, 283, 290, 291, 293, 299, 314, 344, 347, 349, 351, 371, 388, 400, 401, 408, 413, 442, 449, 459, 471, 497, 512, 513, 515, 516, 518, 519, 520, 522, 523, 525, 527, 528, 529, 531, 533, 534, 535, 539, 540, 541, 542, 543, 548, 549, 551, 552, 553, 556, 562, 563, 572, 573, 582, 598, 599, 602, 609, 618, 620, 624, 625, 634, 652, 653, 654, 655, 656, 676, 677, 682, 698, 701, 702, 703, 722, 725, 729, 753, 759, 760, 762, 764, 765, 766, 768, 769, 770, 771, 777, 781, 785, 789, 791, 794, 814, 816, 824, 844, 850, 855, 862, 864, 867, 885, 889, 912, 914, 915, 917, 918, 934, 938, 954, 958, 961, 962, 966, 985, 996, 1004, 1024, 1027, 1030, 1032, 1035, 1051, 1054, 1063, 1066, 1068, 1069, 1070, 1071, 1073, 1074, 1080, 1086, 1100, 1109, 1110, 1111, 1114, 1115, 1126, 1141, 1151, 1153, 1154, 1180, 1201, 1216, 1228, 1244, 1245, 1277, 1279, 1281, 1283, 1286, 1288, 1318, 1337, 1363, 1382, 1406, 1437, 1489, 1499, 1501, 1512, 1513, 1515, 1517, 1520, 1524, 1526, 1527, 1528, 1529, 1531, 1532, 1536, 1539, 1542, 1544, 1546, 1547, 1548, 1549, 1551, 1555, 1561, 1562, 1565, 1568, 1575, 1576, 1579, 1582, 1584, 1597, 1598, 1599, 1600, 1601, 1605, 1607, 1608, 1609, 1610, 1627, 1628, 1637, 1640, 1643, 1645, 1648, 1649, 1652, 1656, 1662, 1665, 1677, 1831, 1832, 1833, 1834, 1835, 1836, 1837 and 1838 indicates nonunion, slow healing or normal healing after acute injury, and/or indicates successful, no or slow healing after a nonunion-mitigating intervention.