U.S. patent application number 13/376046 was filed with the patent office on 2012-06-07 for diagnostic transcriptomic biomarkers in inflammatory cardiomyopathies.
This patent application is currently assigned to University of Miami. Invention is credited to Joshua M. Hare, Bettina Heidecker.
Application Number | 20120142544 13/376046 |
Document ID | / |
Family ID | 43298106 |
Filed Date | 2012-06-07 |
United States Patent
Application |
20120142544 |
Kind Code |
A1 |
Hare; Joshua M. ; et
al. |
June 7, 2012 |
DIAGNOSTIC TRANSCRIPTOMIC BIOMARKERS IN INFLAMMATORY
CARDIOMYOPATHIES
Abstract
Molecular signatures that function as very sensitive diagnostic
biomarker for myocarditis, heart disease and disorders thereof, are
identified.
Inventors: |
Hare; Joshua M.; (Miami
Beach, FL) ; Heidecker; Bettina; (Miami, FL) |
Assignee: |
University of Miami
Miami
FL
|
Family ID: |
43298106 |
Appl. No.: |
13/376046 |
Filed: |
June 2, 2010 |
PCT Filed: |
June 2, 2010 |
PCT NO: |
PCT/US10/37018 |
371 Date: |
February 24, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61183306 |
Jun 2, 2009 |
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Current U.S.
Class: |
506/7 ;
435/320.1; 435/325; 435/6.12; 435/7.92; 436/501; 506/16; 506/18;
530/350; 536/23.1 |
Current CPC
Class: |
C12Q 2600/136 20130101;
C12Q 1/6883 20130101; C12Q 2600/112 20130101; C12Q 2600/158
20130101 |
Class at
Publication: |
506/7 ; 435/6.12;
435/7.92; 435/325; 435/320.1; 436/501; 506/16; 506/18; 530/350;
536/23.1 |
International
Class: |
C40B 30/00 20060101
C40B030/00; G01N 33/566 20060101 G01N033/566; C12N 5/10 20060101
C12N005/10; C07H 21/00 20060101 C07H021/00; C40B 40/06 20060101
C40B040/06; C40B 40/10 20060101 C40B040/10; C07K 14/00 20060101
C07K014/00; C12Q 1/68 20060101 C12Q001/68; C12N 15/63 20060101
C12N015/63 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with U.S. government support under
grant numbers U54-HL081028 (Specialized Center for Cell Based
Therapy) and R01s HL084275, AG025017, HL065455, and HL094849, which
were awarded by the National Institutes of Health. The U.S.
government may have certain rights in the invention,
Claims
1. A transcriptomic biomarker for the identifying patients at risk
of developing myocarditis or idiopathic dilated cardiomyopathy, or
the diagnosis and differentiation between myocarditis and
idiopathic dilated cardiomyopathy comprising one or more marker
signatures, wherein the marker signatures are set forth as: marker
signature I: (1552302_at) FLJ77644.TMEM106, (1552553_a_at) NLRC4,
(1552584_at) IL12RB1, (1554899_s_at) FCER1G, (1555349_a_at) ITGB2,
(1559584_a_at) C16orf54, hCG.sub.--1644884, (1563245_at) MGST1,
(1565162_s_at) ANXA2, (1568126_at) SPP1, (1568574_x_at) IFI30,
(201442_at) CTSC, (201487_at) LAPTM5, (201721_s_at) CD14,
(201743_at) CAPG, (201850_at) PLTP, (202075_s_at) VAMP8,
(202546_at) LYN, (202625_at) ITGB2, (202803_s_at) PCK2, (202847_at)
CSF1R, (203104_at) RASSF2, (203185_at) RPS6KA1, (203379_at) CD53,
(203416_at) PLEK, (203471_s_at) SEMA4D, (203528_at) CD163,
(203645_s_at) PLA2G2A, (203649_s_at) CXCL9, (20391_s_at) CYBB,
(203923_s_at) IRF8, (204057_at) CD48, (204118_at) TYROBP,
(204122_at) GLIPR1, (204222_s_at) FCER1G, (204232_at) PLEKHO2,
(204436_at) CD44, (204490_s_at) SLC7A7, (204588_s_at) STC1,
(204595_s_at) CD52, (204661_at) VSIG4, (204787_at) IL10RA,
(204912_at) SASH3, (204923_at) TLR2, (204924_at) CSTA, (20497 l_at)
CCR1, (205098_at, 205269_at) LCP2, (205270_s_at) GZMA, (205488_at)
CD86, (205685_at) CD8A, (205758_at) ITGAM, (205786_s_at) LY86,
(205859_at) PTPN6, (206687_s_at) CCR2, FLJ78302, (206978_at) PTPRC,
(207238_s_at) SYK, (207540_s_at) LILRB2, (207697_x_at) LCP1,
(208885_at) CORO1A, (209083_at) HLA-DQB1, (209480_at) DLK1,
(209560_s_at) CD44, (209835_x_at) SPP1, (209875_s_at) AIF1,
(209901_x_at) C3AR1, (209906_at) CD300A, (209933_s_at) NCF2,
(209949_at) LILRB2, (210146_x_at) TLR1, (210176_at) LAIR1,
(210644_s_at) LILRB1, (211336_x_at) TRBC1, TRBC2, TRBV19;
(211796_s_at) CD44, (212063_at) PTPRC, (212587_s_at, 212588_at)
HLA-DQA1 HLA-DQA2; (212671_s_at) hCG.sub.--1998957, HLA-DQB1/B2,
HLA-DRB1/2/3/4/5; (21299_x_at) AIF1, (213095_x_at) DOCK2,
(213160_at) HSPA6, (213418_at) RNASE6, (213566_at) RAC2,
(213603_s_at) MYO1F, (213733_at) HLA-DQA1, (213831_at) LYZ,
(213975_s_at) LOC648998, (214084_x_at) CD163, (215049_x_at) AIF1,
(215051_x_at) ADA, (216705_s_at) FCGR1A, FCGR1C; (216950_s_at)
GLUL, (217202_s_at) SNX10, (218404_at) MAFB, (218559_s_at)
CCDC109B, (218802_at) BIN2, (219191_s_at) DOCK10, (219279_at)
SLAMF8, (219386_s_at) SIGLEC1, (219519_s_at) 1-Mar, (219574_at)
MS4A4A, (219607_s_at) MS4A6A, (219666_at) GAL3ST4, (219815_at)
PSTPIP2, (219938_s_at) TLR7, (220146_at) COTL1, (221059_s_at) NPL,
(221210_s_at) SH3BGRL3, (221269_s_at) PYCARD, (221666_s_at) CLEC7A,
(221698_s_at) OBFC2A, (222872_x_at) CENTA2, (222876_s_at,
223343_at) MS4A7, (223344_s_at, 223343_at) MS4A6A, (224356_x_at)
MS4A4A, (224357_s_at) COTL1, (224583_at) BCAT1, (225285_at) C1QC,
(225353_s_at) CTSC, (225646_at) CTSC, (225647_s_at) BCAT1,
(226517_at, 226818_at) MPEG1, (226841_at) FYB, (227266_s_at)
RILPL2, (227983_at) OSR1, (228399_at) C1 orf162, (228532_at)
LILRB1, (230741_at) MRO, (231358_at) CTSS, (232617_at) DOCK8,
(232843_s_at) OBFC2A, (233085_s_at) PARVG, (234987_at) CPM,
(235019_at) HAVCR2, (235458_at) CCL18, (32128_at) CD52, (34210_at)
MAFF, (36711_at) SIGLEC1; or marker signature: (1552411_at)
DEFB106A/B, (1556721_at) FLJ33706, (1559224_at) LCE1E, (1562256 at,
1562257_x_at) NLRP1, (1562785_at) HERC6, (1564281_at) LOC285708,
(1564362_x_at) ZNF843, (1569568_at) NA, (1569569_x_at) NA,
(213609_s_at) SEZ6L, (213791_at) PENK, (224209_s_at) GDA,
(231628_s_at) NA, (243909_x_at) GUSBL2, (244891_x_at) NA; or marker
signature II: (1552411_at) DEFB106A/B, (1556721_at) FLJ33706,
(1559224_at) LCE1E, (1562256_at, 1562257_x_at) NLRP1, (1562785_at)
HERC6, (1564281_at) LOC285708, (1564362_x_at) ZNF843, (1569568_at)
NA, (1569569_x_at) NA, (213609_s_at) SEZ6L, (213791_at) PENK,
(224209_s_at) GDA, (231628_s_at) NA, (243909_x_at) GUSBL2,
(244891_x_at) NA; or, marker signature III: MafB, MafF, MHC class
II, CD44, BCAT1 (Homo sapiens); CCR2, BCAT1, ADA, Annexin II,
Pleckstrin (Homo sapiens); p17-phox, CCR2, p67-phox, Pleckstrin,
IL-12 receptor (Homo sapiens); C1q, CD44, CD14, SLAP-130(ADAP),
alpha-4/beta-1 integrin (Homo sapiens); Plastin, IRT-1 (Homo
sapiens); CD163, HP/HB complex (Homo sapiens); Complement component
C1, Complement C4=Complement component C4a.sup.+, Complement
component C4b, Complement C2=Complement component C2a.sup.+,
Complement component C2b, PLTP, ABCA1, CREB1, Cholesterol
extracellular region,
Cholesterol+ATP+H.sub.2O=Cholesterol+ADP+PO.sub.4.sup.3- (Homo
sapiens); or, marker signature IV: (156328_at) NA, (204477_at)
RABIF, (205275_at) GTPBP1, (214313_s_at) EIF5B; or, marker
signature V: (1552302_at) FLJ77644, TMEM106A; (1552310_at)
C15orf40, (1553212_at) KRT78, (1555349_a_at) ITGB2, (1555878_at)
RPS24, (1556033_at) NA, (1556507_at) NA, (1558605_at) NA
(1559224_at) LCE1E, (1562785_at) HERC6, (1565662_at) NA, (15658300
NA, (202375_at) SEC24D, (202445_s_at) NOTCH2, (203741_s_at) ADCY7,
(204222_s_at) GLIPR1, (206052_s_at) SLBP, (206333_at) MSI1,
(206770_s_at) SLC35A3, (209307_at) SWAP70, (211089_s_at) NEK3,
(211341_at) LOC100131317, POU4F1; (212511_at) PICALM, (212830_at)
MEGF9, (212999_x_at) hCG.sub.--1998957, HLA-DQB1/2,
HLA-DRB1/2/3/4/5; (213501_at) ACOX1, (213831_at) HLA-DQA1,
(217054_at) NA, (217182_at) MUC5AC, (217322_x_at) NA, (217777_s_at)
PTPLAD1, (218803_at) CHFR, (219425_at) SULT4A1, (221663_x_at) HRH3,
(223077_at) TMOD3, (224327_s_at) DGAT2, (224996_at) Na, (225579_at)
PQLC3, (226240_at) MGC21874, (227280_s_at) CCNYL1, (227618_at) Na,
(227983_at) RILPL2, (228980_at) RFFL, (229191_at) TBCD, (230836_at)
ST8SIA4, (231599_x_at) DPF1, (234495_at) KLK15, (234986_at) NA,
(234987_at) NA, (236232_at) STX4, (236404_at) NA, (236698_at) NA,
(238327_at) LOC440836, (238445_x_at) MGAT5B, (239463_at) NA,
(242383_at) NA, (242563_at) NA, (243819_at) NA, (244841_at) SEC24A,
(32069_at) N4BP1, (44673_at) SIGLEC1, (53720_at) C19orf66; or,
marker signature VI: MSI1 (1556507_at), KRT78, KRT78 (1556507_at),
KRT78 (1556507_at), 1556507_at; combinations, complementary
sequences, fragments, alleles, variants, or gene products
thereof.
2. The transcriptomic marker of claim 1, wherein nucleic acids or
peptides thereof, in the marker signature are differentially
expressed as compared to controls.
3. A transcriptomic biomarker for the diagnosis between giant cell
myocarditis and idiopathic dilated cardiomyopathy comprising a
marker signature set forth as: (210667_at) AQP4, (221212_x_at)
PBRM1, (227145_at) LOXL4, (228329_at) DAB1, (231577_s_at) GBP1,
(231906_at) HOXD8, (235334_at) ST6GALNAC3, (237783_at) PLAC8L1,
complementary sequences, fragments, alleles, variants and gene
products thereof.
4. The transcriptomic marker of claim 3, wherein nucleic acids or
peptides thereof, in the marker signature are differentially
expressed as compared to controls.
5. A transcriptomic biomarker for the diagnosis between sarcoidosis
and idiopathic dilated cardiomyopathy comprising a marker signature
set forth as: (1552974_at) NA, (1553781_at) ZC3HAV1L,
(1554478_a_at) HEATR3, (1556760_a_at) NA, (1556883_a_at) LOC440896,
(1557717_at) LOC338862, (1560144-at) NA, (1560683_at) BCL8,
(1560684_x_at) BCL8, (1561543_at) NA, (1562035_at) NA, (1563054_at)
NA, (1563452_at) KIAA0241, (1564107_at) NA, (1564733_at) NA,
(1565788_at) NA, (1566550_at) NA, (1568589_at) NA, (201291_s_at)
TOP2A, (204666_s_at) RP5-1000E10.4, (208356_s_at) BCL2L11,
(209371_s_at) SH3BP2, (215512_at) 6-Mar, (216947_a) DES,
(217292_at) MTMR7, (218554_s_at) ASH1L, (218585_s_at) DTL,
(219528_at) TIPIN (219735_s_at) TFCP2L1, (219918_s_at) ASPM,
(220085_at) HELLS, (220735_s_at) SENP7, (220930_s_at) MGC5590,
(221212_x_at) PBRM1, (221268_s_at) SGPP1, (221969_at) NA,
(223700_at) MND1 (223865_at) SOX6, (224424_x_at) LOC440888,
(224426_s_at) LOC440888, (232453_at) NA, (233786_at) NA,
(235588_at) ESCO2, (235661_at) NA, (235899_at) CA13, (236628_at)
NA, (236470_at) NA, (237289_at) CREB1, (238370_x_at) RPL22,
(238375_at, 239486_at) NA, (239899_at) RNF145, (241922_at) NA,
(242784_at) NA, (242939_at) TFDP1, (244356_at) NA, (244609_at) NA,
(37892_at) COL11A1, complementary sequences, fragments, alleles,
variants and gene products thereof.
6. The transcriptomic marker of claim 5, wherein nucleic acids or
peptides thereof, in the marker signature are differentially
expressed as compared to controls.
7. A transcriptomic biomarker for the diagnosis between peripartum
cardiomyopathy and idiopathic dilated cardiomyopathy comprising a
marker signature set forth as: (1553972_a_at) CBS, (1557833_at) NA,
(1560395_at) NA; (201909_at) LOC100133662, RPS4Y1; (204409_s_at,
204410_at) EIF1AY, (205000_at, 205001_s_at) DDX3Y; (205033_s_at)
DEFA1, DEFA3, LOC728358; (205048_s_at) PSPH, (205609_at) ANGPT1,
(206624_at) LOC100130216, USP9Y; (206700_s_at) JARID1D, (207063_at)
CYorf14, (208067_x_at) LOC100130224, UTY; (209771_x_at) CD24,
(211018_at) LSS, (211149_at) LOC100130224, UTY; (212768_s_at)
OLFM4, (212816_s_at) CBS, (212906_at) GRAMD1B, (214131_at)
CYorf15B, (214218_s_at) XIST, (214983_at) TTTY15, (216758_at) NA,
(219938_s_at) PSTPIP2, (221728_x_at) XIST, (223645_s_at,
223646_s_at) CYorf15B, (224293_at) TTTY10, (224588_at, 224589_at,
224590_at, 227671_at) XIST, (227742_at) CLIC6, (228194_s_at)
SORCS1, (228492_at) LOC100130216, USP9Y; (221960_at) MUM1L1,
(229534_at) ACOT4, (230104_s_at) TPPP, (230760_at) LOC100130829,
ZFY; (231592_at) TSIX, (232365_at) SIAH1, (232618_at) CYorf15A,
(233176_at) NA, (235334_at) ST6GALNAC3, (235446_at) NA, (235942_at)
LOC401629, LOC401630, (236694_at) CYorf15A, (239568_at) PLEKHH2,
(239584_at) NA, (239677_at) NA, (24316_at) NA, (243610_at)
C9orf135, (244482_at) Na, (226_s_at) CD24, complementary sequences,
fragments, alleles, variants and gene products thereof.
8. The transcriptomic marker of claim 7, wherein nucleic acids or
peptides thereof, in the marker signature are differentially
expressed as compared to controls.
9. A transcriptomic biomarker for the diagnosis between systemic
lupus erythematosus and idiopathic dilated cardiomyopathy
comprising a marker signature set forth as: (1552946_at) ZNF114,
(1553607_at) C21orf109, (1555485_s_at) FAM153B, (1558882_at)
LOC401233, (1561012_at) NA, (1566518_at) NA, (1569539_at) NA,
(1569794_at) NA, (207781_s_at) ZNF711, (222375_at) NA, (229288_at)
NA, (229523_at) TTMA, (235803_at) NA, (238553_at) EPHA7,
(238755_at) NA, (240783_at) NA, (240903_at) NA, (242641_at) NA,
(243012_at) NA, (244626_at) NA, (244636_at) NA, complementary
sequences, fragments, alleles, variants and gene products
thereof.
10. The transcriptomic marker of claim 9, wherein nucleic acids or
peptides thereof, in the marker signature are differentially
expressed as compared to controls.
11. A transcriptomic biomarker for the diagnosis between giant cell
myocarditis and lymphocytic myocarditis comprising the marker
signature set forth as: (156328_at) NA, (204477_at) RABIF,
(205275_at) GTPBP1, (214313_s_at) EIF5B, complementary sequences,
fragments, alleles, variants and gene products thereof.
12. The transcriptomic marker of claim 11, wherein nucleic acids or
peptides thereof, in the marker signature are differentially
expressed as compared to controls.
13. A transcriptomic biomarker for the diagnosis between
sarcoidosis and lymphocytic myocarditis comprising a marker
signature set forth as: (20447_at) RABIF, (205275_at) GTPBP1,
(214313_s_at) EIF5B, (224500_s_at) MON1A, (236093_at) NA,
(243564_at) PDE1C, complementary sequences, fragments, alleles,
variants and gene products thereof.
14. The transcriptomic marker of claim 13, wherein nucleic acids or
peptides thereof, in the marker signature are differentially
expressed as compared to controls.
15. A transcriptomic biomarker for the diagnosis between peripartum
cardiomyopathy and lymphocytic myocarditis comprising a marker
signature set forth as: (156328_at) NA, (205275_at) GTPBP1,
(207300_s_at) F7, (214313_s_at) EIF5B, (214473_x_at) PMS2L3,
(227509_x_at) NA, (228232_s_at) VSIG2, (230731_x_at) ZDHHC8,
(232586_x_at) LOC100133315, (236093_at) NA, (237867_s_at) PID1,
(243564_at) PDE1C, complementary sequences, fragments, alleles,
variants and gene products thereof.
16. The transcriptomic marker of claim 15, wherein nucleic acids or
peptides thereof, in the marker signature are differentially
expressed as compared to controls.
17. A transcriptomic biomarker for the diagnosis between systemic
lupus erythematosus and lymphocytic myocarditis comprising a marker
signature set forth as: (1556205_at) NA, (202179_at) BLMH,
(203134_at) PICALM, (203540_at) GFAP, (205554_s_at) DNASE1L3,
(205673_s_at) ASB9, (205794_s_at) NOVA1, (209220_at) GPC3,
(209304_x_at) GADD45B, (209540_at) IGF1, (209923_s_at) BRAP,
(212173_at) AK2, (213469_at) LPPR4 (214338_at) DNAJB12,
(216269_s_at) ELN, (217950_at) NOSIP, (218180_s_at) EPS8L2,
(220117_at) ZNF385D, (220941_s_at) C21orf91, (222002_at) C7orf26,
(222879_s_at) POLH, (223574_x_at) PPP2R2c, (223586_at) ARNTL2,
(230974_at) DDX19B, (233298_at) C13orf38, SOHLH2; (238151_at) NA,
(243076_x_at) GLI4, complementary sequences, fragments, alleles,
variants and gene products thereof.
18. The transcriptomic marker of claim 17, wherein nucleic acids or
peptides thereof, in the marker signature are differentially
expressed as compared to controls.
19. A transcriptomic biomarker for the differential diagnosis
between giant cell myocarditis and sarcoidosis comprising a marker
signature set forth as: (1553894_at) CCDC122, (1557311_at)
LOC100131354, (1557996_at) POLR2J4, (1558430_at) NA, (1559227_s_at)
VHL, (1561789_at) NA, (1569312_at) NA, (205238_at) CXorf34,
(211734_s_at) FCER1A, (218699_at) RAP2C, (225207_at) PDK4,
(231114_at) SPATA22, (231418_at) NA, (231819_at) NA, (231956_at)
KIAA1618, (233927_at) NA, (239151_at) CTGLF6, (241788_x_at) NA,
(242691_at) NA, complementary sequences, fragments, alleles,
variants and gene products thereof.
20. The transcriptomic marker of claim 19, wherein nucleic acids or
peptides thereof, in the marker signature are differentially
expressed as compared to controls.
21. A transcriptomic biomarker for the diagnosis of myocarditis
comprising a marker signature set forth as: (1552302_at) FLJ77644,
TMEM106A; (1552310_at) C15orf40, (1553212_at) KRT78, (1555349_a_at)
ITGB2, (1555878_at) RPS24, (1556033_at) NA, (1556507_at) NA,
(1558605_at) NA (1559224_at) LCE1E, (1562785_at) HERC6,
(1565662_at) NA, (1565830_at) NA, (202375_at) SEC24D, (202445_s_at)
NOTCH2, (203741_s_at) ADCY7, (204222_s_at) GLIPR1, (206052_s_at)
SLBP, (206333_at) MSI1, (206770_s_at) SLC35A3, (209307_at) SWAP70,
(211089_s_at) NEK3, (211341_at) LOC100131317, POU4F1; (212511_at)
PICALM, (212830_at) MEGF9, (212999_x_at) hCG.sub.--1998957,
HLA-DQB1/2, HLA-DRB1/2/3/4/5 (213501_at) ACOX1, (213831_at)
HLA-DQA1, (217054_at) NA, (217182_at) MUC5AC, (217322_x_at) NA,
(217777_s_at) PTPLAD1, (218803_at) CHFR, (219425_at) SULT4A1,
(221663_x_at) HRH3, (223077.sub.2A) TMOD3, (224327_s_at) DGAT2,
(224996_at) Na, (225579_at) PQLC3, (226240_at) MGC21874,
(227280_s_at) CCNYL1, (227618_at) Na, (227983_at) RILPL2,
(228980_at) RFFL, (229191_at) TBCD, (230836_at) ST8SIA4,
(231599_x_at) DPF1, (234495_at) KLK15, (234986_at) NA, (234987_at)
NA, (236232_at) STX4, (236401 at NA, (236698_at) NA, (238327_at)
LOC440836, (238445_x_at) MGAT5B, (239463_at) NA, (242383_at) NA,
(242563_at) NA, (243819_at) NA, (244841_at) SEC24A, (32069_at)
N4BP1, (44673_at) SIGLEC1, (53720_at) C19orf66, complementary
sequences, fragments, alleles, variants and gene products
thereof.
22. The transcriptomic marker of claim 21, wherein nucleic acids or
peptides thereof, in the marker signature are differentially
expressed as compared to controls.
23. A transcriptomic biomarker for the diagnosis of myocarditis
versus idiopathic dilated cardiomyopathy comprising a marker
signature set forth as: MSI1 (1556507_at), KRT78, KRT78
(1556507_at), KRT78 (1556507_at), 1556507_at, complementary
sequences, fragments, alleles, variants and gene products
thereof.
24. The transcriptomic marker of claim 23, wherein nucleic acids or
peptides thereof, in the marker signature are differentially
expressed as compared to controls.
25. A transcriptomic biomarker for the diagnosis of subtypes of
inflammatory cardiomyopathy versus idiopathic dilated
cardiomyopathy comprising marker signatures set forth in Table 18,
complementary sequences, fragments, alleles, variants and gene
products thereof.
26. A transcriptomic biomarker for the diagnosis of rare types of
inflammatory cardiomyopathy versus lymphocytic myocarditis
comprising one or more marker signatures set forth in Tables 1 to
19, complementary sequences, fragments, alleles, variants and gene
products thereof.
27. A transcriptomic biomarker for the diagnosis of patients at
risk of developing one or more cardiac disorders comprising one or
more marker signatures set forth as: marker signature I:
(1552302_at) FLJ77644, TMEM106, (1552553_a_at) NLRC4, (1552584_at)
IL12R131, (1554899_s_at) FCER1G, (1555349_a_at) ITGB2,
(1559584_a_at) C16orf54, hCG.sub.--1644884, (1563245_at) MGST1,
(1565162_s_at) ANXA2, (1568126_at) SPP1, (1568574_x_at) IFI30,
(201442_at) CTSC, (201487_at) LAPTM5, (201721_s_at) CD14,
(201743_at) CAPG, (201850_at) PLTP, (202075_s_at) VAMP8,
(202546_at) LYN, (202625_at) ITGB2, (202803_s_at) PCK2, (202847_at)
CSF1R, (203104_at) RASSF2, (203185_at) RPS6KA1, (203379_at) CD53,
(203416_at) PLEK, (203471 is at) SEMA4D, (203528_at) CD163,
(203645_s_at) PLA2G2A, (203649_s_at) CXCL9, (203915_at) CYBB,
(203923_s_at) IRF8, (204057_at) CD48, (204118_at) TYROBP,
(204122_at) GLIPR1, (204222_s_at) FCER1G, (204232_at) PLEKHO2,
(204436_at) CD44, (204490_s_at) SLC7A7, (204588_s_at) STC1,
(204595_s_at) CD52, (204661_at) VSIG4, (204787_at) IL10RA,
(204912_at) SASH3, (204923_at) TLR2, (204924_at) CSTA, (204971_at)
CCR1, (205098_at, 205269_at) LCP2, (205270_s_at) GZMA, (205488_at)
CD86, (205685_at) CD8A, (205758_at) ITGAM, (205786_s_at) LY86,
(205859_at) PTPN6, (206687_s_at) CCR2, FLJ78302, (206978_at) PTPRC,
(207238_s_at) SYK, (207540_s_at) LILRB2, (207697_x_at) LCP1,
(208885_at) CORO1A, (209083_at) HLA-DQB1, (209480_at) DLK1,
(209560_s_at) CD44, (209835_x_at) SPP1, (209875_s_at) A1F1,
(209901_x_at) C3AR1, (209906_at) CD300A, (209933_s_at) NCF2,
(209949_at) LILRB2, (210146_x_at) TLR1, (210176_at) LAIR1,
(210644_s_at) LILRB1, (211336_x_at) TRBC1, TRBC2, TRBV19;
(211796_s_at) CD44, (212063_at) PTPRC, (212587_s_at, 212588_at)
HLA-DQA1 HLA-DQA2; (212671_s_at) hCG.sub.--1998957, HLA-DQB1/B2,
HLA-DRB1/2/3/4/5; (21299_x_at) AIF1, (213095_x_at) DOCK2,
(213160_at) HSPA6, (213418_at) RNASE6, (213566_at) RAC2,
(213603_s_at) MYO1F, (213733_at) HLA-DQA1, (213831_at) LYZ,
(213975_s_at) LOC648998, (214084_x_at) CD163, (215049_x_at) AIF1,
(215051_x_at) ADA, (216705_s_at) FCGR1A, FCGR1C; (216950_s_at)
GLUL, (217202_s_at) SNX10, (218404_at) MAFB, (218559_s_at)
CCDC109B, (218802_at) BIN2, (219191_s_at) DOCK10, (219279_at)
SLAMF8, (219386_s_at) SIGLEC1, (219519_s_at) 1-Mar, (219574_at)
MS4A4A, (219607_s_at) MS4A6A, (219666_at) GAL3ST4, (219815_at)
PSTPIP2, (219938_s_at) TLR7, (220146_at) COTL1, (221059_s_at) NPL,
(221210_s_at) SH3BGRL3, (221269_s_at) PYCARD, (221666_s_at) CLEC7A,
(221698_s_at) OBFC2A, (222872_x_at) CENTA2, (222876_s_at,
223343_at) MS4A7, (223344_s_at, 223343_at) MS4A6A, (224356_x_at)
MS4A4A, (224357_s_at) COTL1, (224583_at) BCAT1, (225285_at) C1QC,
(225353_s_at) CTSC, (225646_at) CTSC, (225647_s_at) BCAT1,
(226517_at, 226818_at) MPEG1, (226841_at) FYB, (227266_s_at)
RILPL2, (227983_at) OSR1, (228399_at) C1orf162, (228532_at) LILRB1,
(230741_at) MRO, (231358_at) CTSS, (232617_at) DOCK8, (232843_s_at)
OBFC2A, (233085_s_at) PARVG, (234987_at) CPM, (235019_at) HAVCR2,
(235458_at) CCL18, (32128_at) CD52, (34210_at) MAFF, (36711_at)
SIGLEC1; marker signature II: (1552411_at) DEFB106A/B, (1556721_at)
FLJ33706, (1559224_at) LCE1E, (1562256_at, 1562257_x_at) NLRP1,
(1562785_at) HERC6, (1564281_at) LOC285708, (1564362_x_at) ZNF843,
(1569568_at) NA, (1569569_x_at) NA, (213609_s_at) SEZ6L,
(213791_at) PENK, (224209_s_at) GDA, (231628_s_at) NA,
(243909_x_at) GUSBL2, (244891_x_at) NA; marker signature III: MafB,
MafF, MHC class II, CD44, BCAT1 (Homo sapiens); CCR2, BCAT1, ADA,
Annexin II, Pleckstrin (Homo sapiens); p47-phox, CCR2, p67-phox,
Pleckstrin, IL-12 receptor (Homo sapiens); C1q, CD44, CD14,
SLAP-130(ADAP), alpha-4/beta-1 integrin (Homo sapiens); Plastin,
IRT-1 (Homo sapiens); CD163, HP/HB complex (Homo sapiens);
Complement component C1, Complement C4=Complement component
C4a.sup.+, Complement component C4b, Complement C2=Complement
component C2a.sup.+, Complement component C2b, PLTP, ABCA1, CREB1,
Cholesterol extracellular region,
Cholesterol+ATP+H.sub.2O=Cholesterol+ADP+PO.sub.4.sup.3- (Homo
sapiens); marker signature IV: (210667_at) AQP4, (221212_x_at)
PBRM1, (227145_at) LOXL4, (228329_at) DAB1, (231577_s_at) GBP1,
(231906_at) HOXD8, (235334_at) ST6GALNAC3, (237783_at) PLAC8L1;
marker signature V: (1552974_at) NA, (1553781_at) ZC3HAV1L,
(1554478_a_at) HEATR3, (1556760_a_at) NA, (1556883_a_at) LOC440896,
(1557717_at) LOC338862, (1560144-at) NA, (1560683_at) BCL8,
(1560684_x_at) BCL8, (1561543_at) NA, (1562035_at) NA, (1563054_at)
NA, (1563452_at) K1AA0241, (1564107_at) NA, (1564733_at) NA,
(1565788_at) NA, (1566550_at) NA, (1568589_at) NA, (201291_s_at)
TOP2A, (204666_s_at) RP5-1000E10.4, (208356_s_at) BCL2L11,
(209371_s_at) SH3BP2, (215512_at) 6-Mar, (216947_at) DES,
(217292_at) MTMR7, (218554_s_at) ASH1L, (218585_s_at) DTL,
(219528_at) TIPIN (219735_s_at) TFCP2L1, (219918_s_at) ASPM,
(220085_at) HELLS, (220735_s_at) SENP7, (220930_s_at) MGC5590,
(221212_x_at) PBRM1, (221268_s_at) SGPP1, (221969_at) NA,
(223700_at) MND1, (223865_at) SOX6, (224424_x_at) LOC440888,
(224426_s_at) LOC440888, (232453_at) NA, (233786_at) NA,
(235588_at) ESCO2, (235661_at) NA, (2358990 CA13, (236628_at) NA,
(236470_at) NA, (237289_at) CREB1, (238370_x_at) RPL22, (238375_at,
239486_at) NA, (239899_at) RNF145, (241922_at) NA, (242784_at) NA,
(242939_at) TFDP1, (244356_at) NA, (244609_at) NA, (37892_at)
COL11A1, marker signature VI: (1553972_a_at) CBS, (1557833_at) NA,
(1560395_at) NA; (201909_at) LOC100133662, RPS4Y1; (204409_s_at,
204410_at) EIF1AY, (205000_at, 205001_s_at) DDX3Y; (205033_s_at)
DEFA1, DEFA3, LOC728358; (205048_s_at) PSPH, (205609_at) ANGPT1,
(206624_at) LOC100130216, USP9Y; (206700_s_at) JARID1D, (207063_at)
CYorf14, (208067_x_at) LOC100130224, UTY; (209771_x_at) CD24,
(211018_at) LSS, (211149_at) LOC100130224, UTY; (212768_s_at)
OLFM4, (212816_s_at) CBS, (212906_at) GRAMD1B, (214131_at)
CYorf15B, (214218_s_at) XIST, (214983_at) TTTY15, (216758_at) NA,
(219938_s_at) PSTPIP2, (221728_x_at) XIST, (223645_s_at,
223646_s_at) CYorf15B, (224293_at) TTTY10, (224588_at, 224589_at,
224590_at, 227671_at) XIST, (227742_at) CLIC6, (228194_s_at)
SORCS1, (228492_at) LOC100130216, USP9Y; (221960_at) MUM1L1,
(229534_at) ACOT4, (230104_s_at) TPPP, (230760_at) LOC100130829,
ZFY; (231592_at) TSIX, (232365_at) SIAH1, (232618_at) CYorf15A,
(233176_at) NA, (235334_at) ST6GALNAC3, (235446_at) NA, (235942_at)
LOC401629, LOC401630, (236694_at) CYorf15A, (239568_at) PLEKHH2,
(239584_at) NA, (239677_at) NA, (24316_at) NA, (243610_at)
C9orf135, (244482_at) Na, (226_s_at) CD24; marker signature VII:
(1552946_at) ZNF114, (1553607_at) C21orf109, (1555485_s_at)
FAM153B, (1558882_at) LOC401233, (1561012_at) NA, (1566518_at) NA,
(1569539_at) NA, (1569794_at) NA, (207781_s_at) ZNF711, (222375_at)
NA, (229288_at) NA, (229523_at) TTMA, (235803_at) NA, (238553_at)
EPHA7, (238755_at) NA, (240783_at) NA, (240903_at) NA, (242641_at)
NA, (243012_at) NA, (244626_at) NA, (244636_at) NA; marker
signature VIII: (156328_at) NA, (204477_at) RABIF, (20275_at)
GTPBP1, (214313_s_at) EIF5B; marker signature IX: (20447_at) RABIF,
(205275_at) GTPBP1, (214313_s_at) EIF5B, (224500_s_at) MON1A,
(236093_at) NA, (243564_at) PDE1C; marker signature X: (156328_at)
NA, (205275_at) GTPBP1, (207300_s_at) F7, (214313_s_at) EIF5B,
(214473_x_at) PMS2L3, (227509_x_at) NA, (228232_s_at) VSIG2,
(230731_x_at) ZDHHC8, (232586_x_at) LOC100133315, (236093_at) NA,
(237867_s_at) PID1, (243564_at) PDE1C; marker signature XI:
(1556205_at) NA, (202179_at) BLMH, (203134_at) PICALM, (203540_at)
GFAP, (205554_s_at) DNASE1L3, (205673_s_at) ASB9, (205794_s_at)
NOVA1, (209220_at) GPC3, (209304_x_at) GADD45B, (209540_at) IGF1,
(209923_s_at) BRAP, (212173_at) AK2, (213469_at) LPPR4 (214338_at)
DNAJB12, (216269_s_at) ELN, (217950_at) NOSIP, (218180_s_at)
EPS8L2, (220117_at) ZNF385D, (220941_s_at) C21orf91, (222002_at)
C7orf26, (222879_s_at) POLH, (223574_x_at) PPP2R2C, (223586_at)
ARNTL2, (230974_at) DDX19B, (233298_at) C13orf38, SOHLH2;
(238151_at) NA, (243076_x_at) GLI4; marker signature XII:
(1553894_at) CCDC122, (1557311_at) LOC100131354, (1557996_at)
POLR2J4, (1558430_at) NA, (1559227_s_at) VHL, (1561789_at) NA,
(1569312_at) NA, (205238_at) CXorf34, (211734_s_at) FCER1A,
(218699_at) RAP2C, (225207_at) PDK4, (231114_at) SPATA22,
(231418_at) NA, (231819_at) NA, (231956_at) KIAA1618, (233927_at)
NA, (239151_at) CTGLF6, (241788_x_at) NA, (242691_at) NA; marker
signature XIII: (1552302_at) FLJ77644, TMEM106A; (1552310_at)
C15orf40, (1553212_at) KRT78, (1555349_a_at) ITGB2, (1555878_at)
RPS24, (1556033_at) NA, (1556507_at) NA, (1558605_at) NA, (1559221
at) LCE1E, (1562785_at) HERC6, (1565662_at) NA, (1565830_at) NA,
(202375_at) SEC24D, (202445_s_at) NOTCH2, (203741_s_at) ADCY7,
(204222_s_at) GLIPR1, (206052_s_at) SLBP, (206333_at) MSI1,
(206770_s_at) SLC35A3, (209307_at) SWAP70, (211089_s_at) NEK3,
(211341_at) LOC100131317, POU4F1; (212511_at) PICALM, (212830_at)
MEGF9, (212999_x_at) hCG.sub.--1998957, HLA-DQB1/2,
HLA-DRB1/2/3/4/5; (213501_at) ACOX1, (213831_at) HLA-DQA1,
(217054_at) NA, (217182_at) MUC5AC, (217322_x_at) NA, (217777_s_at)
PTPLAD1, (218803_at) CHFR, (219425_at) SULT4A1, (221663_x_at) HRH3,
(223077_at) TMOD3, (224327_s_at) DGAT2, (224996_at) Na, (225579_at)
PQLC3, (226240_at) MGC21874, (227280_s_at) CCNYL1, (227618_at) Na,
(227983_at) RILPL2, (228980_at) RFFL, (229191_at) TBCD, (230836_at)
ST8SIA4, (231599_x_at) DPF1, (234495_at) KLK15, (234986_at) NA,
(234987_at) NA, (236232_at) STX4, (236404_at) NA, (236698_at) NA,
(238327_at) LOC440836, (238445_x_at) MGAT5B, (239463_at) NA,
(242383_at) NA, (242563_at) NA, (243819_at) NA, (244841_at) SEC24A,
(32069_at) N4BP1, (44673_at) SIGLEC1, (53720_at) C19orf66; marker
signature XIV: MSI1 (1556507_at), KRT78, KRT78 (1556507_at), KRT78
(1556507_at), 1556507_at; marker signature XV: (201721_s_at) CD14,
(1554899_s_at) FCER1G, (210146_x_at) TLR1, (204923_at) TLR2,
(1555349_a_at) ITGB2, (44673_at) SIGLEC1, (219938_s_at) TLR7,
(203741_s_at) ADCY7, (212830_at) MEGF9, (21777_s_at) PTPLAD1,
(209307_at) SWAP70, (206333_at) MSI1, (1559224_at) LCE1E; or
combinations thereof.
28. A transcriptomic biomarker for diagnosing one or more cardiac
diseases or disorders comprising one or more marker signatures of
claim 26.
29. A method of diagnosing myocarditis and other cardiac diseases
or disorders, comprising: identifying in a biological sample from a
patient a molecular signature set forth in Tables 1 to 19,
complementary sequences, fragments, alleles, variants and gene
products thereof; assessing the probability of identification of
each component in each sample; assigning each to a class; and,
diagnosing myocarditis and other cardiac disorders.
30. A method of diagnosing heart disease or myocarditis comprising:
identifying in a biological sample from a patient a molecular
signature set forth in Tables 1 to 19, complementary sequences,
fragments, alleles, variants and gene products thereof; assessing
the probability of identification of each component gene in each
sample; assigning each to a class; and, diagnosing heart disease or
myocarditis.
31. A kit comprising a transcriptomic biomarker of any one or more
molecular signatures set forth in Tables 1 to 19.
32. A cell expressing any one or more biomolecules selected from
Tables 1 to 19.
33. A vector encoding any one or more biomolecules selected from
Tables 1 to 19.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the priority of U.S.
provisional patent application No. 61/183,306 filed Jun. 2, 2009,
which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] This invention relates to biomarkers of heart disease,
myocarditis, novel drug therapeutic targets, compositions and
methods of predicting, diagnosing and treating heart diseases and
related disorders thereof. More specifically, the invention
concerns methods and compositions based on unique molecular
signatures associated with various aspects of cardiac diseases and
disorders.
BACKGROUND
[0004] The myocardites are inflammatory diseases of the heart that
have variable clinical presentations and are caused by a range of
underlying inflammatory variants. Of new onset heart failure,
10-30% may be caused by cardiac inflammation, and viral infection
systemic or local inflammatory diseases, or genetic predisposition
represent inciting factors. Myocarditis can be difficult to
diagnose requiring multiple endomyocardial biopsies (EMBs). Even
with multiple biopsies, consensus among pathologists has been
difficult to attain. Inaccurate or uncertain diagnosis is of major
concern, since emerging therapies specifically targeting
inflammatory or viral heart disease, have the potential to reverse
the disease process. In a previous decision analysis investigating
the value of EMBs to improve clinical outcome with specific
therapy, histological inaccuracy was a major limiting factor for
efficacy of treatment. In addition, the important subtypes of
myocarditis have highly distinct outcomes, require markedly
different therapeutic strategies, and can be difficult to
distinguish based on standard histopathology. Current attempts to
improve diagnostic accuracy include screening for viral RNA in
endomyocardial biopsies, serum anti-heart autoantibodies, and use
of magnetic resonance imaging (MRI).
SUMMARY
[0005] Molecular signatures that function as very sensitive
diagnostic biomarker for myocarditis, cardiovascular diseases and
disorders, heart disease and disorders thereof, were identified.
The biomarkers also distinguish between various cardiac diseases
and disorders allowing for accurate diagnosis. In addition the
biomarkers provide for the identification of individuals at risk of
developing cardiac diseases and disorders. The transcriptomic
biomarkers provide for the early diagnosis of cardiovascular
diseases or disorders.
[0006] Transcriptomic biomarkers (TBBs) were identified to
distinguish or differentially diagnose between giant cell
myocarditis and cardiac sarcoidosis; peripartum cardiomyopathy and
lymphocytic cardiomyopathy; myocarditis and idiopathic dilated
cardiomyopathy; cardiac sarcoidosis, giant cell myocarditis,
peripartum cardiomyopathy, and systemic lupus erythematosus with
cardiac involvement. The biomarkers or marker signatures comprised
molecules some of which were up-regulated, down-regulated, no
change, absent, etc (i.e., differentially expressed) as compared to
normal healthy controls. The signatures not only allow for the
early diagnosis and diagnostic differentiation between various
diseases and disorders hut also for identifying individuals at risk
for one or more cardiovascular diseases or disorders.
[0007] Other aspects of the invention are described infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows: Significance Analysis of Microarrays Plot of
differentially expressed genes in lymphcytic myocarditis vs
idiopathic dilated cardiomyopathy: There were 9,878 genes
differentially expressed in myocardits (n=16) vs IDCM (n=32;
q<5%; fold change>1.2), of which 2,313 were overexpressed
(depicted in red) and 7,565 were downregulated (depicted in
green).
[0009] FIG. 2: Validation of a 62-gene molecular signature in an
independent test set (idiopathic dilated cardiomyopathy: n=10,
myocarditis: n=5) using Prediction Analysis of Microarrays (PAM):
The y-ordinate illustrates the predicted test probability values
obtained from PAM analysis; x-ordinate lists the number of samples.
While samples were assigned to different classes with varying
probability values, the classification accuracy of the
transcriptomic biomarker was 100%.
[0010] FIG. 3: Distinction of patients with idiopathic dilated
cardiomyopathy vs lymphocytic myocarditis based on results from
quantitative realtime RT-PCR: This heatmap was created with an
unsupervised clustering approach based on Euclidean distance in R,
using the detected gene expression levels from quantitative
realtime RT-PCR as confirmatory test. Columns represent samples and
rows represent genes labeled with their corresponding gene symbol.
Application of the developed 13 genes molecular signature through
realtime RT-PCR correctly identified all samples.
[0011] FIGS. 4A-4B: Principal Components Analysis (PCA) of patients
with myocarditis vs idiopathic dilated cardiomyopathy (IDCM): To
illustrate significance of each of the 62 genes thr phenotypic
categorization, PCA was performed with correlation matrix in
samples from patients with myocarditis (n=16) or IDCM (n=32) with
genes as variables. Genes are labeled with serial numbers and
expression levels of each individual gene are illustrated as Eigen
vector towards the class, in which they are overexpressed. Vectors
close to the center with close to vertical direction depict genes
that were less robust, while genes that were highly specific for a
phenotype were illustrated as vectors with endpoint distant from
the center directing towards the corresponding clustered set of
samples of a specific phenotype, A) Clustered samples from patients
with myocarditis are labeled "M", while IDCM samples are labeled
"I". All samples from myocarditis, except two, were noticeably
grouped together, suggesting that a small set of 62 genes enables
clear distinction between patients with inflammatory heart disease
and IDCM. Importantly, those two samples were also misclassified
the heatmap analysis, while Prediction Analysis of Microarrays
identified both of them correctly. B) Encircled are genes that were
repeatedly identified to be the most robust markers of myocarditis,
when various algorithms of Misclassified-Penalized Posterior
classification were applied. Output from PCA places those genes
both far from the center as well as distant from the vertical line,
confirming that these are highly robust classifiers for
myocarditis.
DETAILED DESCRIPTION
[0012] The present invention is described with reference to the
attached figures, wherein like reference numerals are used
throughout the figures to designate similar or equivalent elements.
The figures are not drawn to scale and they are provided merely to
illustrate the instant invention. Several aspects of the invention
are described below with reference to example applications for
illustration. It should be understood that numerous specific
details, relationships, and methods are set forth to provide a full
understanding of the invention. One having ordinary skill in the
relevant art, however, will readily recognize that the invention
can be practiced without one or more of the specific details or
with other methods. The present invention is not limited by the
illustrated ordering of acts or events, as some acts may occur in
different orders and/or concurrently with other acts or events.
Furthermore, not all illustrated acts or events are required to
implement a methodology in accordance with the present
invention.
[0013] All genes, gene names, and gene products disclosed herein
are intended to correspond to homologs from any species for which
the compositions and methods disclosed herein are applicable. Thus,
the terms include, but are not limited to genes and gene products
from humans and mice. It is understood that when a gene or gene
product from a particular species is disclosed, this disclosure is
intended to be exemplary only, and is not to be interpreted as a
limitation unless the context in which it appears clearly
indicates. Thus, for example, for the genes disclosed herein, which
in some embodiments relate to mammalian nucleic acid and amino acid
sequences are intended to encompass homologous and/or orthologous
genes and gene products from other animals including, but not
limited to other mammals, fish, amphibians, reptiles, and birds, in
preferred embodiments, the genes or nucleic acid sequences are
human.
[0014] Unless otherwise defined, all terms including technical and
scientific terms) used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. It will be further understood that terms, such
as those defined in commonly used dictionaries, should be
interpreted as having a meaning that is consistent with their
meaning in the context of the relevant art and will not be
interpreted in an idealized or overly formal sense unless expressly
so defined herein.
DEFINITIONS
[0015] In accordance with the present invention and as used herein,
the following terms are defined with the following meanings, unless
explicitly stated otherwise.
[0016] As used herein, "a", "an," and "the" include plural
references unless the context clearly dictates otherwise.
[0017] As used herein, a "molecular signature" or "signature" or
"biomarker" or "transcriptomic based biomarker" are used
interchangeably herein and refers to the biomolecules identified in
Tables 1 to 19. Thus, Table 1 comprising the biomolecules listed
therein, represents one biomarker or molecular signature; Table 2
comprising the biomolecules listed therein, represents another one
biomarker or molecular signature; and so forth. As more
biomolecules are discovered, each newly identified biomolecules can
be assigned to any one or more biomarker or molecular signature.
Each biomolecule can also be removed, reassigned or reallocated to
a molecular signature. Thus, in some embodiments the molecular
signature comprises at least ten biomolecules. The ten biomolecules
are selected from the genes identified herein, or from newly
identified biomolecules. Any one of the signatures can be used in
the diagnosis of a disease or disorder, for example, myocarditis
and idiopathic cardiomyopathy or differentiate between myocarditis
and idiopathic cardiomyopathy. Mammalian sequences are preferred,
with human sequences the most preferred.
[0018] The term "biomolecule" refers to DNA, RNA (including mRNA,
rRNA, tRNA and tmRNA), nucleotides, nucleosides, analogs,
polynucleotides, peptides and any combinations thereof.
[0019] A base "position" as used herein refers to the location of a
given base or nucleotide residue within a nucleic acid.
[0020] As used herein, the term "array" refers to an ordered
spatial arrangement, particularly an arrangement of immobilized
biomolecules.
[0021] As used herein, the term "addressable array" refers to an
array wherein the individual elements have precisely defined x and
y coordinates, so that a given element at a particular position in
the array can be identified.
[0022] As used herein, the terms "probe" and "biomolecular probe"
refer to a biomolecule used to detect a complementary biomolecule.
Examples include antigens that detect antibodies, oligonucleotides
that detect complimentary oligonucleotides, and ligands that detect
receptors. Such probes are preferably immobilized on a
microelectrode comprising a substrate.
[0023] As used herein, the terms "bioarray," "biochip" and "biochip
array" refer to an ordered spatial arrangement of immobilized
biomolecules on a microelectrode arrayed on a solid supporting
substrate. Preferred probe molecules include aptamers, nucleic
acids, oligonucleotides, peptides, ligands, antibodies and
antigens; peptides and proteins are the most preferred probe
species. Biochips, as used in the art, encompass substrates
containing arrays or microarrays, preferably ordered arrays and
most preferably ordered, addressable arrays, of biological
molecules that comprise one member of a biological binding pair.
Typically, such arrays are oligonucleotide arrays comprising a
nucleotide sequence that is complementary to at least one sequence
that may be or is expected to be present in a biological sample.
Alternatively, and preferably, proteins, peptides or other small
molecules can be arrayed in such biochips for performing, inter
alia, immunological analyses (wherein the arrayed molecules are
antigens) or assaying biological receptors (wherein the arrayed
molecules are ligands, agonists or antagonists of said
receptors).
[0024] Expression/amount of a gene, biomolecule, or biomarker in a
first sample is at a level "greater than" the level in a second
sample if the expression level/amount of the gene or biomarker in
the first sample is at least about 1 time, 1.2 times, 1.5 times,
1.75 times, 2 times, 3 times 4 times, 5 times, 6 times, 7 times, 8
times, 9 times, 10 times, 20 times, 30 times, the expression
level/amount of the gene or biomarker in the second sample or a
normal sample. Expression levels/amounts can be determined based on
any suitable criterion known in the art, including but not limited
to mRNA, cDNA, proteins, protein fragments and/or gene copy.
Expression levels/amounts can be determined qualitatively and/or
quantitatively.
[0025] By the term "modulate," it is meant that any of the
mentioned activities, are, e.g., increased, enhanced, increased,
agonized (acts as an agonist), promoted, decreased, reduced,
suppressed blocked, or antagonized (acts as an agonist). Modulation
can increase activity more than 1-fold, 2-fold, 3-fold, 5-fold,
10-fold, 100-fold, etc., over baseline values. Modulation can also
decrease its activity below baseline values.
[0026] An "allele" or "variant" is an alternative form of a gene.
Variants may result from at least one mutation in the nucleic acid
sequence and may result in altered mRNAs or in polypeptides whose
structure or function may or may not be altered. Any given natural
or recombinant gene may have none, one, or many allelic forms.
Common mutational changes that give rise to variants are generally
ascribed to natural deletions, additions, or substitutions of
nucleotides. Each of these types of changes may occur alone, or in
combination with the others, one or more times in a given
sequence.
[0027] The term, "complementary" means that two sequences are
complementary when the sequence of one can bind to the sequence of
the other in an anti-parallel sense wherein the 3'-end of each
sequence binds to the 5'-end of the other sequence and each A,
T(U), G, and C of one sequence is then aligned with a T(U), A, C,
and G, respectively, of the other sequence. Normally, the
complementary sequence of the oligonucleotide has at least 80% or
90%, preferably 95%, most preferably 100%, complementarity to a
defined sequence. Preferably, alleles or variants thereof can be
identified. A BLAST program also can be employed to assess such
sequence identity.
[0028] The term "complementary sequence" as it refers to a
polynucleotide sequence, relates to the base sequence in another
nucleic acid molecule by the base-pairing rules. More particularly,
the term or like term refers to the hybridization or base pairing
between nucleotides or nucleic acids, such as, for instance,
between the two strands of a double stranded DNA molecule or
between an oligonucleotide primer and a primer binding site on a
single stranded nucleic acid to be sequenced or amplified.
Complementary nucleotides are, generally, A and T (or A and U), or
C and G. Two single stranded RNA or DNA molecules are said to be
substantially complementary when the nucleotides of one strand,
optimally aligned and compared and with appropriate nucleotide
insertions or deletions, pair with at least about 95% of the
nucleotides of the other strand, usually at least about 98%, and
more preferably from about 99% to about 100%. Complementary
polynucleotide sequences can be identified by a variety of
approaches including use of well-known computer algorithms and
software, for example the BLAST program.
[0029] As used herein, the term "aptamer" or "selected nucleic acid
binding species" shall include non-modified or chemically modified
RNA or DNA. The method of selection may be by, but is not limited
to, affinity chromatography and the method of amplification by
reverse transcription (RT) or polymerase chain reaction (PCR).
[0030] As used herein, the term "signaling aptamer" shall include
aptamers with reporter molecules, preferably a fluorescent dye,
appended to a nucleotide in such a way that upon conformational
changes resulting from the aptamer's interaction with a ligand, the
reporter molecules yields a differential signal, preferably a
change in fluorescence intensity.
[0031] As used herein, the term "fragment or segment", as applied
to a nucleic acid sequence, gene or polypeptide, will ordinarily be
at least about 5 contiguous nucleic acid bases (for nucleic acid
sequence or gene) or amino acids (for polypeptides), typically at
least about 10 contiguous nucleic acid bases or amino acids, more
typically at least about 20 contiguous nucleic acid bases or amino
acids, usually at least about 30 contiguous nucleic acid bases or
amino acids, preferably at least about 40 contiguous nucleic acid
bases or amino acids; more preferably at least about 50 contiguous
nucleic acid bases or amino acids, and even more preferably at
least about 60 to 80 or more contiguous nucleic acid bases or amino
acids in length, "Overlapping fragments" as used herein, refer to
contiguous nucleic acid or peptide fragments which begin at the
amino terminal end of a nucleic acid or protein and end at the
carboxy terminal end of the nucleic acid or protein. Each nucleic
acid or peptide fragment has at least about one contiguous nucleic
acid or amino acid position in common with the next nucleic acid or
peptide fragment, more preferably at least about three contiguous
nucleic acid bases or amino acid positions in common, most
preferably at least about ten contiguous nucleic acid bases amino
acid positions in common.
[0032] "Biological samples" include solid and body fluid samples.
Preferably, the sample is obtained from heart. However, the
biological samples used in the present invention can include cells,
protein or membrane extracts of cells, blood or biological fluids
such as ascites fluid or brain fluid (e.g., cerebrospinal fluid).
Examples of solid biological samples include, but are not limited
to, samples taken from tissues of the central nervous system, bone,
breast, kidney, cervix, endometrium, head/neck, gallbladder,
parotid gland, prostate, pituitary gland, muscle, esophagus,
stomach, small intestine, colon, liver, spleen, pancreas, thyroid,
heart, lung, bladder, adipose, lymph node, uterus, ovary, adrenal
gland, testes, tonsils and thymus. Examples of "body fluid samples"
include, but are not limited to blood, serum, semen, prostate
fluid, seminal fluid, urine, saliva, sputum, mucus, bone marrow,
lymph, and tears.
[0033] "Sample" is used herein in its broadest sense. A sample
comprising polynucleotides, polypeptides, peptides, antibodies and
the like may comprise a bodily fluid; a soluble fraction of a cell
preparation, or media in which cells were grown; a chromosome, an
organelle, or membrane isolated or extracted from a cell; genomic
DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound
to a substrate; a cell; a tissue; a tissue print; a fingerprint,
skin or hair; and the like.
[0034] "Diagnostic" means identifying the presence or nature of a
pathologic condition. Diagnostic methods differ in their
sensitivity and specificity. The "sensitivity" of a diagnostic
assay is the percentage of diseased individuals who test positive
(percent of "true positives"). Diseased individuals not detected by
the assay are "false negatives." Subjects who are not diseased and
who test negative in the assay, are termed "true negatives." The
"specificity" of a diagnostic assay is 1 minus the false positive
rate, where the "false positive" rate is defined as the proportion
of those without the disease who test positive. While a particular
diagnostic method may not provide a definitive diagnosis of a
condition, it suffices if the method provides a positive indication
that aids in diagnosis.
Transcriptomic Biomarker/Molecular Signatures
[0035] The invention comprises molecular signatures that function
as very sensitive diagnostic biomarkers for heart failure, heart
diseases, myocarditis, and other heart disorders. These biomarkers
also identify individuals at risk of developing cardiovascular
diseases or disorders. Myocarditis is a common disease that is
estimated to cause up to 30% of dilated, cardiomyopathy, even in
patients initially asymptomatic. Myocarditis can also present as
sudden cardiac death and affects individuals of all ages. In
childhood, myocarditis causes a greater percentage of heart failure
than in adulthood. The fact that the majority of viral induced
cases pass in a clinically unapparent course, points out the
significance of finding more reliable biomarkers than standard
diagnostic tools which are currently available, e.g. ECG, cardiac
enzymes and immunohistochemistry.
[0036] Transcriptomics have emerged as a highly valuable tool to
aid in complex pathologic diagnosis. A transcriptome was used to
create biomarkers (TBBs) that add diagnostic accuracy to clinical,
pathological and imaging modalities currently used to diagnose
myocarditis.
[0037] Derails of the experimental procedures are provided in the
examples section which follows. Briefly, a microarray analysis was
performed in a case-control fashion on samples from patients with
histologically proven myocarditis (n=16) and idiopathic dilated
cardiomyopathy (IDCM, n=32) to develop highly accurate diagnostic
transcriptomic biomarkers using multiple classification algorithms.
Additional gene signatures were obtained to distinguish between
cardiac sarcoidosis (n=9), giant cell myocarditis (n=3), peripartum
cardiomyopathy (n=6), and systemic lupus erythematosus with cardiac
involvement (n=3).
[0038] 9,878 genes were identified and which were differentially
expressed in lymphocytic myocarditis vs. IDCM (FC>1.2,
FDR<5%), from which a transcriptomic biomarker containing 62
genes was identified, which distinguished myocarditis with 100%
sensitivity (95% CI: 46-100%) and 100% specificity (95% CI:
66-100%). Multiple classification algorithms and quantitative
realtime RT-PCR analysis further reduced this subset to a highly
robust molecular signature of 13 genes, which still performed with
100% accuracy. TBBs were also obtained to distinguish between giant
cell myocarditis and cardiac sarcoidosis, and peripartum
cardiomyopathy vs lymphocytic cardiomyopathy.
[0039] Transcriptomic biomarkers can improve the clinical detection
of patients with inflammatory diseases of the heart. This approach
advances the clinical management and treatment of cardiac disorders
with highly variable outcome.
[0040] In preferred embodiments, diagnosis to distinguish between
giant cell myocarditis and cardiac sarcoidosis; peripartum
cardiomyopathy vs lymphocytic cardiomyopathy; myocarditis and
idiopathic dilated cardiomyopathy; cardiac sarcoidosis, giant cell
myocarditis, peripartum cardiomyopathy, and systemic lupus
erythematosus with cardiac involvement, comprises identifying a
marker signature set forth in any one of Tables 1 to 19,
complementary sequences, fragments, alleles, variants and gene
products thereof.
[0041] For example, a transcriptomic biomarker comprises a
molecular signature such as for example: marker signature I:
(1552302_at) FLJ77644.TMEM106, (1552553_a_at) NLRC4, (1552584_at)
IL12RB1, (1554899_s_at) FCER1G, (1555349_a_at) ITGB2,
(1559584_a_at) C16orf54, hCG.sub.--1644884, (1563245_at) MGST1,
(1565162_s_at) ANXA2, (1568126_at) SPP1, (1568574_x_at) IFI30,
(201442_at) CTSC, (201487_at) LAPTM5, (201721_s_at) CD14,
(201743_at) CAPG, (201850_at) PLTP, (202075_s_at) VAMP8,
(202546_at) LYN, (202625_at) ITGB2, (202803_s_at) PCK2, (202847_at)
CSF1R, (203104_at) RASSF2, (203185_at) RPS6KA1, (203379_at) CD53,
(203416_at) PLEK, (203471_s_at) SEMA4D, (203528_at) CD163,
(203645_s_at) PLA2G2A, (203649_s_at) CXCL9, (203915_at) CYBB,
(203923_s_at) IRF8, (204057_at) CD48, (204118_at) TYROBP,
(204122_at) GLIPR1, (204222_s_at) FCER1G, (204232_at) PLEKHO2,
(204436_at) CD44, (204490_s_at) SLC7A7, (204588_s_at) STC1,
(204595_s_at) CD52, (204661_at) VSIG4, (204787_at) IL10RA,
(204912_at) SASH3, (204923_at) TLR2, (204924_at) CSTA, (204971_at)
CCR1, (205098_at, 205269_at) LCP2, (205270_s_at) GZMA (205488_at)
CD86, (205685_at) CD8A, (205758_at) ITGAM, (205786_s_at) LY86,
(205859_at) PTPN6, (206687_s_at) CCR2, FLJ78302, (206978_at) PTPRC,
(207238_s_at) SYK, (207540_s_at) LILRB2, (207697_x_at) LCP1,
(208885_at) CORO1A, (209083_at) HLA-DQB1, (209480_at) DLK1,
(209560_s_at) CD44, (209835_x_at) SPP1, (209875_s_at) AIF1,
(209901_x_at) C3AR1, (209906_at) CD300A, (209933_s_at) NCF2,
(209949_at) LILRB2, (210146_x_at) TLR1, (210176_at) LAIR1,
(210644_s_at) LILRB1, (211336_x_at) TRBC1, TRBC2, TRBV19;
(211796_s_at) CD44, (212063_at) PTPRC, (212587_s_at, 212588_at)
HLA-DQA1 HLA-DQA2; (212671_s_at) hCG.sub.--1998957, HLA-DQB1/B2,
HLA-DRB1/2/3/4/5; (21299_x_at) AIF1, (213095_x_at) DOCK2,
(213160_at) HSPA6, (213418_at) RNASE6, (213566_at) RAC2,
(213603_s_at) MYO1F, (213733_at) HLA-DQA1, (213831_at) LYZ,
(213975_s_at) LOC648998, (214084_x_at) CD163, (215049_x_at) AIF1,
(215051_x_at) ADA, (216705_s_at) FCGR1A, FCGR1C; (216950_s_at)
GLUL, (217202_s_at) SNX10, (218404_at) MAFB, (218559_s_at)
CCDC109B, (218802_at) BIN2, (219191_s_at) DOCK10, (219279_at)
SLAMF8, (219386_s_at) SIGLEC1, (219519_s_at) 1-Mar, (219574_at)
MS4A4A, (219607_s_at) MS4A6A, (219666_at) GAL3ST4, (219815_at)
PSTPIP2, (219938_s_at) TLR7, (220146_at) COTL1, (221059_s_at) NPL,
(221210_s_at) SH3BGRL3, (221269_s_at) PYCARD, (221666_s_at) CLEC7A,
(221698_s_at) OBFC2A, (222872_x_at) CENTA2, (222876_s_at,
223343_at) MS4A7, (223344_s_at, 223343_at) MS4A6A, (224356_x_at)
MS4A4A, (224357_s_at) COTL1, (224583_at) BCAT1, (225285_at) C1QC,
(225353_s_at) CTSC, (225646_at) CTSC, (225647_s_at) BCAT1,
(226517_at, 226818_at) MPEG1, (226841_at) FYB, (227266_s_at)
RILPL2, (227983_at) OSR1, (228399_at) C1orf162, (228532_at) LILRB1,
(230741_at) MRO, (231358_at) CTSS, (232617_at) DOCK8, (232843_s_at)
OBFC2A, (233085_s_at) PARVG, (234987_at) CPM, (235019_at) HAVCR2,
(235458_at) CCL18, (32128_at) CD52, (34210_at) MAFF, (36711_at)
SIGLEC1; or marker signature: (1552411_at) DEFB106A/B, (1556721_at)
FLJ33706, (1559224_at) LCE1E, (1562256_at, 1562257_x_at) NLRP1,
(1562785_at) HERC6, (1564281_at) LOC285708, (1564362_x_at) ZNF843,
(1569568_at) NA, (1569569_x_at) NA, (213609_s_at) SEZ6L,
(213791_at) PENK, (224209_s_at) GDA, (231628_s_at) NA,
(243909_x_at) GUSBL2, (244891_x_at) NA; or, marker signature II:
(1552411_at) DEFB106A/B, (1556721_at) FLJ33706, (1559224_at) LCE1E,
(1562256_at, 1562257_x_at) NLRP1, (1562785_at) HERC6, (1564281_at)
LOC285708, (1564362_x_at) ZNF843, (1569568_at) NA, (1569569_x_at)
NA, (213609_s_at) SEZ6L, (213791_at) PENK, (224209_s_at) GDA,
(231628_s_at) NA, (243909_x_at) GUSBL2, (244891_x_at) NA; or,
marker signature III: MafB, MafF, MHC class II, CD44, BCAT1 (Homo
sapiens); CCR2, BCAT1, ADA, Annexin Pleckstrin (Homo sapiens);
p47-phox, CCR2, p67-phox, Pleckstrin, IL-12 receptor (Homo
sapiens); C1q, CD44, CD14, SLAP-130(ADAP), alpha-4/beta-1 integrin
(Homo sapiens); Plastin, IRT-1 (Homo sapiens); CD163, HP/HB complex
(Homo sapiens); Complement component C1, Complement C4=Complement
component C4a.sup.+, Complement component C4b, Complement
C2=Complement component C2a.sup.+, Complement component C2b, PLTP,
ABCA1, CREB1, Cholesterol extracellular region,
Cholesterol+ATP+H.sub.2O=Cholesterol+ADP+PO.sub.4.sup.3- (Homo
sapiens); or, marker signature IV: (156328_at) NA, (204477_at)
RABIF, (205275_at) GTPBP1, (214313_s_at) EIF5B; or, marker
signature V: (1552302_at) FLJ77644, TMEM106A; (1552310_at)
C15orf40, (1553212_at) KRT78, (1555349_a_at) ITGB2, (1555878_at)
RPS24, (1556033_at) NA, (1556507_at) NA, (1558605_at) NA
(1559224_at) LCE1E, (1562785_at) HERC6, (1565662_at) NA,
(1565830_at) NA, (202375_at) SEC24D, (202445_s_at) NOTCH2,
(203741_s_at) ADCY7, (204222_s_at) GLIPR1, (206052_s_at) SLBP,
(206333_at) MSI1, (206770_s_at) SLC35A3, (209307_at) SWAP70,
(211089_s_at) NEK3, (211341_at) LOC100131317, POU4F1; (212511_at)
PICALM, (212830_at) MEGF9, (212999_x_at) hCG.sub.--1998957,
HLA-DQB1/2, HLA-DRB1/2/3/4/5; (213501_at) ACOX1, (213831_at)
HLA-DQA1, (217054_at) NA, (217182_at) MUC5AC, (217322_x_at) NA,
(217777_s_at) PTPLAD1, (218803_at) CHFR, (219425_at) SULT4A1,
(221663_x_at) HRH3, (223077_at) TMOD3, (224327_s_at) DGAT2,
(224996_at) Na, (225579_at) PQLC3, (226240_at) MGC21874,
(227280_s_at) CCNYL1, (227618_at) Na, (227983_at) RILPL2,
(228980_at) RFFL, (229191_at) TBCD, (230836_at) ST8SIA4,
(231599_x_at) DPF1, (234495_at) KLK15, (234986_at) NA, (234987_at)
NA, (236232_at) STX4, (236404_at) NA, (236698_at) NA, (238327_at)
LOC440836, (238445_x_at) MGAT5B, (239463_at) NA, (242383_at) NA,
(242563_at) NA, (243819_at) NA, (244841_at) SEC24A, (32069_at)
N4BP1, (44673_at) SIGLEC1, (53720_at) C19orf66; or, marker
signature VI: MSI1 (1556507_at), KRT78, KRT78 (1556507_at), KRT78
(1556507_at), 1556507_at, Detection of any one or more signatures,
combinations of signatures, complementary sequences, fragments,
alleles, variants, or gene products thereof, comprise a
transcriptomic biomarker.
[0042] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis between giant cell myocarditis and idiopathic
dilated cardiomyopathy comprising a marker signature set forth as:
(210667_at) AQP4, (221212_x_at) PBRM1, (227145_at) LOXL4,
(228329_at) DAB1, (231577_s_at) GBP1, (231906_at) HOXD8,
(235334_at) ST6GALNAC3, (237783_at) PLAC8L1, complementary
sequences, fragments, alleles, variants and gene products
thereof.
[0043] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis between sarcoidosis and idiopathic dilated
cardiomyopathy comprising a marker signature set forth as:
(1552974_at) NA, (1553781_at) ZC3HAV1L, (1554478_a_at) HEATR3,
(1556760_a_at) NA, (1556883_a_at) LOC440896, (1557717_at)
LOC338862, (1560144-at) NA, (1560683_at) BCL8, (1560684_x_at) BCL8,
(1561543_at) NA, (1562035_at) NA, (1563054_at) NA, (1563452_at)
K1AA0241, (1564107_at) NA, (1564733_at) NA, (1565788_at)
(1566550_at) NA, (1568589_at) NA, (201291_s_at) TOP2A,
(204666_s_at) RP5-1000E10.4, (208356_s_at) BCL2L11, (209371_s_at)
SH3BP2, (215512_at) 6-Mar, (216947_at) DES, (217292_at) MTMR7,
(218554_s_at) ASH1L, (218585_s_at) DTL, (219528_at) TIPIN
(219735_s_at) TFCP2L1, (219918_s_at) ASPM, (220085_at) HELLS,
(220735_s_at) SENP7, (220930_s_at) MGC5590, (221212_x_at) PBRM1,
(221268_s_at) SGPP1, (221969_at) NA, (223700_at) MND1, (223865_at)
SOX6, (224424_x_at) LOC440888, (224426_s_at) LOC440888, (232453_at)
NA, (233786_at) NA, (235588_at) ESCO2, (235661_at) NA, (235899_at)
CA13, (236628_at) NA, (236470_at) NA, (237289_at) CREB1,
(238370_x_at) RPL22, (238375_at, 239486_at) NA, (239899_at) RNF145,
(241922_at) NA, (242784_at) NA, (242939_at) TFDP1, (244356_at) NA,
(244609_at) NA, (37892_at) COL11A1, complementary sequences,
fragments, alleles, variants and gene products thereof.
[0044] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis between peripartum cardiomyopathy and idiopathic
dilated cardiomyopathy comprising a marker signature set forth as:
(1553972_a_at) CBS, (1557833_at) NA, (1560395_at) NA; (201909_at)
LOC100133662, RPS4Y1; (204409_s_at, 204410_at) EIF1AY, (205000_at,
205001_s_at) DDX3Y; (205033_s_at) DEFA1, DEFA3, LOC728358,
(205048_s_at) PSPH, (205609_at) ANGPT1, (206624_at) LOC100130216,
USP9Y; (206700_s_at) JARID1D, (207063_at) CYorf14, (208067_x_at)
LOC100130224, UTY; (209771_x_at) CD24, (211018_at) LSS, (211149_at)
LOC100130224, UTY; (212768_s_at) OLFM4, (212816_s_at) CBS,
(212906_at) GRAMD1B, (214131_at) CYorf15B, (214218_s_at) XIST,
(214983_at) TITY15, (216758_at) NA, (219938_s_at) PSTPIP2,
(221728_x_at) XIST, (223645_s_at, 223646_s_at) CYorf15B,
(224293_at) TTTY10, (224588_at, 224589_at, 224590_at, 227671_at)
XIST, (227742_at) CLIC6, (228194_s_at) SORCS1, (228492_at)
LOC100130216, USP9Y; (221960_at) MUM1L1, (229534_at) ACOT4,
(230104_s_at) TPPP, (230760_at) LOC100130829, ZFY; (231592_at)
TSIX, (232365_at) SIAH1, (232618_at) CYorf15A, (233176_at) NA,
(235334_at) ST6GALNAC3, (235446_at) NA, (235942_at) LOC401629,
LOC401630, (236694_at) CYorf15A, (239568_at) PLEKHH2, (239584_at)
NA, (239677_at) NA, (24316_at) NA, (243610_at) C9orf135,
(244482_at) Na, (226_s_at) CD24, complementary sequences,
fragments, alleles, variants and gene products thereof.
[0045] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis between systemic lupus erythematosus and
idiopathic dilated cardiomyopathy comprising a marker signature set
forth as: (1552946_at) ZNF114, (1553607_at) C21orf109,
(1555485_s_at) FAM153B, (1558882_at) LOC401233, (1561012_at) NA,
(156618_at) NA, (1569539_at) NA, (1569794_at) NA, (207781_s_at)
ZNF711, (222375_at) NA, (229288_at) NA, (229523_at) TTMA,
(235803_at) NA, (238553_at) EPHA7, (238755_at) NA, (240783_at) NA,
(240903_at) NA, (242641_at) NA, (243012_at) NA, (2446260_at) NA,
(244636_at) NA, complementary sequences, fragments, alleles,
variants and gene products thereof.
[0046] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis between giant cell myocarditis and lymphocytic
myocarditis comprising the marker signature set forth as:
(156328_at) NA, (204477_at) RABIF, (205275_at) GTPBP1,
(214313_s_at) EIF5B, complementary sequences, fragments, alleles,
variants and gene products thereof.
[0047] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis between sarcoidosis and lymphocytic myocarditis
comprising a marker signature set forth as: (20447_at) RABIF,
(205275_at) GIPBP1, (214313_s_at) EIF5B, (224500_s_at) MON1A,
(236093_at) NA, (243564_at) PDE1C, complementary sequences,
fragments, alleles, variants and gene products thereof.
[0048] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis between peripartum cardiomyopathy and lymphocytic
myocarditis comprising a marker signature set forth as: (156328_at)
NA, (205275_at) GTPBP1, (207300_s_at) F7, (214313_s_at) EIF5B,
(214473_x_at) PMS2L3, (227509_x_at) NA, (228232_s_at) VSIG2,
(230731_x_at) ZDHHC8, (232586_x_at) LOC100133315, (236093_at) NA,
(237867_s_at) PID1, (243564_at) PDE1C, complementary sequences,
fragments, alleles, variants and gene products thereof.
[0049] In another preferred embodiment, a transcriptomic biomarker
thr the diagnosis between systemic lupus erythematosus and
lymphocytic myocarditis comprising a marker signature set forth as:
(1556205_at) NA, (202179_at) BLMH, (203134_at) PICALM, (203540_at)
GFAP, (205554_s_at) DNASE1L3, (205673_s_at) ASB9, (205794_s_at)
NOVA1, (209220_at) GPC3, (209304_x_at) GADD45B, (209540_at) IGF1,
(209923_s_at) BRAP, (212173_at) AK2, (213469_at) LPPR4 (214338_at)
DNAJB12, (216269_s_at) ELN, (217950_at) NOSIP, (218180_s_at)
EPS8L2, (220117_at) ZNF385D, (220941_s_at) C21orf91, (222002_at)
C7orf26, (222879_s_at) POLH, (223574_x_at) PPP2R2C, (223586_at)
ARNTL2, (230974_at) DDX19B, (233298_at) C13orf38, SOHLH2;
(238151_at) NA, (243076_x_at) GLI4, complementary sequences,
fragments, alleles, variants and gene products thereof.
[0050] In another preferred embodiment, a transcriptomic biomarker
for the differential diagnosis between giant cell myocarditis and
sarcoidosis comprising a marker signature set forth as:
(1553894_at) CCDC122, (1557311_at) LOC100131354, (1557996_at)
POLR2J4, (1558430_at) NA, (1559227_s_at) VHL, (1561789_at) NA,
(1569312_at) NA, (205238_at) CXorf34, (211734_s_at) FCER1A,
(218699_at) RAP2C, (225207_at) PDK4, (231114_at) SPATA22,
(231418_at) NA, (231819_at) NA, (231956_at) KIAA1618, (233927_at)
NA, (239151_at) CTGLF6, (241788_x_at) NA, (242691_at) NA,
complementary sequences, fragments, alleles, variants and gene
products thereof.
[0051] in another preferred embodiment, a transcriptomic biomarker
for the diagnosis of myocarditis comprising a marker signature set
forth as: (1552302_at) FLJ77644, TMEM106A; (1552310_at) C15orf40,
(1553212_at) KRT78, (1555349_a_at) ITGB2, (1555878_at) RPS24,
(1556033_at) NA, (1556507_at) NA, (1558605_at) NA (1559224_at)
LCE1E, (1562785_at) HERC6, (1565662_at) NA, (1565830_at) NA,
(202375_at) SEC24D, (202445_s_at) NOTCH2, (203741_s_at) ADCY7,
(204222_s_at) GLIPR1, (206052_s_at) SLBP, (206333_at) MSI1,
(206770_s_at) SLC35A3, (209307_at) SWAP70, (211089_s_at) NEK3,
(211341_at) LOC100131317, POU4F1; (212511_at) PICALM, (212830_at)
MEGF9, (212999_x_at) hCG.sub.--1998957, HLA-DQB1/2,
HLA-DRB1/2/3/4/5; (213501_at) ACOX1, (213831_at) HLA-DQA1,
(217054_at) NA, (217182_at) MUC5AC, (217322_x_at) NA, (217777_s_at)
PTPLAD1, (218803_at) CHFR, (219425_at) SULT4A1, (221663_x_at) HRH3,
(223077_at) TMOD3, (224327_s_at) DGAT2, (224996_at) Na, (225579_at)
PQLC3, (226240_at) MGC21874, (227280_s_at) CCNYL (227618_at) Na,
(227983_at) RILPL2, (228980_at) RFFL, (229191_at) TBCD, (230836_at)
ST8SIA4, (231599_x_at) DPF1, (234495_at) KLK15, (234986_at) NA,
(234987_at) NA, (236232_at) STX4, (236404_at) NA, (236698_at) NA,
(238327_at) LOC440836, (238445_x_at) MGAT5B, (239463_at) NA,
(242383_at) NA, (242563_at) NA, (243819_at) NA, (244841_at) SEC24A,
(32069_at) N4BP1, (44673_at) SIGLEC1, (53720_at) C19orf66,
complementary sequences, fragments, alleles, variants and gene
products thereof.
[0052] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis of myocarditis versus idiopathic dilated
cardiomyopathy comprising a marker signature set forth as: MSI1
(1556507_at), KRT78, KRT78 (1556507_at), KRT78 (1556507_at),
1556507_at, complementary sequences, fragments, alleles, variants
and gene products thereof.
[0053] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis and differential diagnosis between myocarditis
and idiopathic dilated cardiomyopathy comprising the marker
signatures set forth in Tables 1, 2, 3, or 15, complementary
sequences, fragments, alleles, variants and gene products
thereof.
[0054] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis between giant cell myocarditis and idiopathic
dilated cardiomyopathy comprising the marker signatures set forth
in Table 4, complementary sequences, fragments, alleles, variants
and gene products thereof.
[0055] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis between sarcoidosis and idiopathic dilated
cardiomyopathy comprising the marker signature set forth in Table
5, complementary sequences, fragments, alleles, variants and gene
products thereof.
[0056] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis between peripartum cardiomyopathy and idiopathic
dilated cardiomyopathy comprising the marker signature set forth in
Table 6, complementary sequences, fragments, alleles, variants and
acne products thereof.
[0057] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis between systemic lupus erythematosus and
idiopathic dilated cardiomyopathy comprising the marker signature
set forth in Table 7, complementary sequences, fragments, alleles,
variants and gene products thereof.
[0058] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis between giant cell myocarditis and lymphocytic
myocarditis comprising the marker signature set forth in Table 8,
complementary sequences, fragments, alleles, variants and gene
products thereof.
[0059] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis between sarcoidosis and lymphocytic myocarditis
comprising the marker signature set forth in Table 9, complementary
sequences, fragments, alleles, variants and gene products
thereof.
[0060] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis between peripartum cardiomyopathy and lymphocytic
myocarditis comprising the marker signature set forth in Table 10,
complementary sequences, fragments, alleles, variants and acne
products thereof.
[0061] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis between systemic lupus erythematosus and
lymphocytic myocarditis comprising the marker signature set forth
in Table 11, complementary sequences, fragments, alleles, variants
and gene products thereof.
[0062] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis between giant cell myocarditis and sarcoidosis
comprising the marker signature set forth in Table 12,
complementary sequences, fragments, alleles, variants and acne
products thereof.
[0063] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis of myocarditis comprising the marker signature
set forth in Table 14, complementary sequences, fragments, alleles,
variants and gene products thereof.
[0064] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis of subtypes of inflammatory cardiomyopathy vs
idiopathic dilated cardiomyopathy comprising the marker signatures
set forth in Table 18, complementary sequences, fragments, alleles,
variants and gene products thereof.
[0065] In another preferred embodiment, a transcriptomic biomarker
for the diagnosis of rare types of inflammatory cardiomyopathy
lymphocytic myocarditis comprising the marker signatures set forth
in Table 19, complementary sequences, fragments, alleles, variants
and gene products thereof.
[0066] In another preferred embodiment, comprises an antibody or
aptamer specific for each gene sequence set froth in Tables 1 to
19, complementary sequences, fragments, alleles, variants and gene
products thereof, complementary sequences, fragments, alleles,
variants and gene products thereof.
[0067] In another preferred embodiment, a biochip comprising
nucleic acid sequences set forth in Tables 1 to 19, complementary
sequences, fragments, alleles, variants and gene products
thereof.
[0068] A method of diagnosing myocarditis and other cardiac
disorders, comprising: identifying in a biological sample from a
patient a molecular signature set forth in Tables 1 to 19,
complementary sequences, fragments, alleles, variants and gene
products thereof; assessing the probability of identification of
each component gene in each sample; assigning each to a class; and,
diagnosing myocarditis and other cardiac disorders.
[0069] In another preferred embodiment, a method of diagnosing
heart disease or myocarditis comprising: identifying in a
biological sample from a patient a molecular signature set forth in
Tables 1 to 19, complementary sequences, fragments, alleles,
variants and gene products thereof; assessing the probability of
identification of each component gene in each sample; assigning
each to a class; and, diagnosing heart disease or myocarditis.
[0070] In another preferred embodiment, a kit comprising a
transcriptomic biomarker of any one or more molecular signatures
set forth in Tables 1 to 19.
[0071] In another preferred embodiment, a cell expressing any one
or more biomolecules selected from Tables 1 to 19.
[0072] In another preferred embodiment, a vector encoding any one
or more biomolecules selected from Tables 1 to 19.
[0073] In another preferred embodiment, the detection in a cell or
patient of the biomolecules, complementary sequences, fragments,
alleles, variants and gene products thereof, is diagnostic of
myocarditis, idiopathic cardiomyopathy, heart, diseases and
disorders thereof. Preferably, the biomolecule sequences,
complementary sequences, fragments, alleles, variants and gene
products thereof, are modulated at levels by at least between 1%,
2%, 5%, 10% in a cell or patient as compared to levels in a normal
cell or normal subject; more preferably, the gene biomarker
sequences, complementary sequences, fragments, alleles, variants
and gene products thereof, are modulated by about 50% in a cell or
a patient as compared to levels in a normal cell or normal subject;
more preferably, the gene biomarker sequences, complementary
sequences, fragments, alleles, variants and gene products thereof,
are modulated by about 75% in a cell or a patient as compared to
levels in a normal cell or normal subject. The term "modulated"
refers to an increase or decrease in level, concentration, amount
etc, as compared to a normal cell or normal healthy subject. The
term can also be applied as "differential expression" wherein one
or more markers are increased, decreased or remain at baseline
levels relative to each other and baseline normal controls.
Alternative Methods and Materials for Identifying Molecular
Signatures or Transcriptomic Biomarkers
[0074] Detection of Nucleic Acids and Proteins as Markers: In
preferred embodiments, each biomarker is detected on chip based
methods such as those described in detail in the examples which
follow. In order to provide accurate diagnosis of cardiac disorders
and diseases, for example, heart failure, myocarditis, idiopathic
cardiomyopathy and the like. Other methods are also known in the
art and one or more methods can be utilized.
[0075] The methods and assays disclosed herein are directed to the
examination of expression of transcriptomic biomarkers in a
mammalian tissue or cell sample, wherein the determination of that
expression of one or more such transcriptomic biomarkers is
predictive of prognostic outcome or diagnostic of cardiac and
cardiovascular diseases and disorders, such as for example,
myocarditis, Coronary Heart Disease, angina, Acute Coronary
Syndrome, Aortic Aneurysm and Dissection, arrhythmias,
Cardiomyopathy, Congenital Heart Disease, congestive heart failure
or chronic heart failure, pericarditis, and the like. The Molecular
signatures or Transcriptomic biomarker comprise the biomolecules
identified in Tables 1 to 19.
[0076] Preferred embodiments in the identification of biomolecules,
analytical methods etc, are described in detail in the Examples
which follow.
[0077] Microarrays: In general, using nucleic acid microarrays,
test and control mRNA samples from test and control tissue samples
are reverse transcribed and labeled to generate cDNA probes. The
probes are then hybridized to an array of nucleic acids immobilized
on a solid support. The array is configured such that the sequence
and position of each member of the array is known. For example, a
selection of genes that have potential to be expressed in certain
disease states may be arrayed on a solid support. Hybridization of
a labeled probe with a particular array member indicates that the
sample from which the probe was derived expresses that gene.
Differential gene expression analysis of disease tissue can provide
valuable information. Microarray technology utilizes nucleic acid
hybridization techniques and computing technology to evaluate the
mRNA expression profile of thousands of genes within a single
experiment. (see, e.g., WO 01/75166 published Oct. 11, 2001; (See,
for example, U.S. Pat. No. 5,700,637, U.S. Pat. No. 5,445,934, and
U.S. Pat. No. 5,807,522, Lockart, Nature Biotechnology,
14:1675-1680 (1996); Cheung, V. G. et al., Nature Genetics
21(Suppl):15-19 (1999) for a discussion of array fabrication). DNA
microarrays are miniature arrays containing gene fragments that are
either synthesized directly onto or spotted onto glass or other
substrates. Thousands of genes are usually represented in a single
array. A typical microarray experiment involves the following
steps: 1) preparation of fluorescently labeled target from RNA
isolated from the sample, 2) hybridization of the labeled target to
the microarray, 3) washing, staining, and scanning of the array, 4)
analysis of the scanned image and 5) generation of gene expression
profiles. Currently two main types of DNA microarrays are being
used: oligonucleotide (usually 25 to 70 mers) arrays and gene
expression arrays containing PCR products prepared from cDNAs. In
forming an array, oligonucleotides can be either prefabricated and
spotted to the surface or directly synthesized on to the surface
(in situ). The Affymetrix GENECHIP.TM. system is a commercially
available microarray system which comprises arrays fabricated by
direct synthesis of oligonucleotides on a glass surface.
[0078] Probe/Gene Arrays: Oligonucleotides, usually 25 mers, are
directly synthesized onto a glass wafer by a combination of
semiconductor-based photolithography and solid phase chemical
synthesis technologies. Each array contains up to 400,000 different
oligonucleotides and each oligonucleotide is present in millions of
copies. Since oligonucleotide probes are synthesized in known
locations on the array, the hybridization patterns and signal
intensities can be interpreted in terms of gene identity and
relative expression levels by the Affymetrix Microarray Suite
software. Each gene is represented on the array by a series of
different oligonucleotide probes. Each probe pair consists of a
perfect match oligonucleotide and a mismatch oligonucleotide. The
perfect match probe has a sequence exactly complimentary to the
particular gene and thus measures the expression of the gene. The
mismatch probe differs from the perfect match probe by a single
base substitution at the center base position, disturbing the
binding of the target gene transcript. This helps to determine the
background and nonspecific hybridization that contributes to the
signal measured for the perfect match oligonucleotide. The
Microarray Suite software subtracts the hybridization intensities
of the mismatch probes from those of the perfect match probes to
determine the absolute or specific intensity value for each probe
set. Probes are chosen based on current information from GenBank
and other nucleotide repositories. The sequences are believed to
recognize unique regions of the 3' end of the gene. A GeneChip
Hybridization Oven ("rotisserie" oven) is used to carry out the
hybridization of up to 64 arrays at one time. The fluidics station
performs washing and staining of the probe arrays. It is completely
automated and contains four modules, with each module holding one
probe array. Each module is controlled independently through
Microarray Suite software using preprogrammed fluidics protocols.
The scanner is a confocal laser fluorescence scanner which measures
fluorescence intensity emitted by the labeled cRNA bound to the
probe arrays. The computer workstation with Microarray Suite
software controls the fluidics station and the scanner. Microarray
Suite software can control up to eight fluidics stations using
preprogrammed hybridization, wash, and stain protocols for the
probe array. The software also acquires and converts hybridization
intensity data into a presence/absence call for each gene using
appropriate algorithms. Finally, the software detects changes in
gene expression between experiments by comparison analysis and
formats the output into .txt files, which can be used with other
software programs for further data analysis.
[0079] The expression of a selected biomarker may also be assessed
by examining gene deletion or gene amplification. Gene deletion or
amplification may be measured by any one of a wide variety of
protocols known in the art, for example, by conventional Southern
blotting, Northern blotting to quantitate the transcription of mRNA
(Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)), dot
blotting (DNA analysis), or in situ hybridization (e.g., FISH),
using an appropriately labeled probe, cytogenetic methods or
comparative genomic hybridization (CGH) using an appropriately
labeled probe.
[0080] Detection of Polypeptides: In another embodiment of the
present invention, a polypeptide corresponding to a marker is
detected. A preferred agent for detecting a polypeptide of the
invention is an antibody or aptamer capable of binding to a
polypeptide corresponding to a marker of the invention, preferably
an antibody with a detectable label. Antibodies can be polyclonal,
or more preferably, monoclonal. An intact antibody, or a fragment
thereof e.g., Fab or F(ab').sub.2 can be used. The term "labeled",
with regard to the probe or antibody, is intended to encompass
direct-labeling of the probe or antibody by coupling, i.e.,
physically linking, a detectable substance to the probe or
antibody, as well as indirect-labeling of the probe or antibody by
reactivity with another reagent that is directly-labeled. Examples
of indirect labeling include detection of a primary antibody using
a fluorescently-labeled secondary antibody and end-labeling of a
DNA probe with biotin such that it can be detected with
fluorescently-labeled streptavidin.
[0081] Proteins from individuals can be isolated using techniques
that are well-known to those of skill in the art. The protein
isolation methods employed can, e.g., be such as those described in
Harlow & Lane (1988), supra. A variety of formats can be
employed to determine whether a sample contains a protein that
binds to a given antibody. Expression of various biomarkers in a
sample can be analyzed by a number of methodologies, many of which
are known in the art and understood by the skilled artisan,
including but not limited to, immunohistochemical and/or Western
analysis, quantitative blood based assays (as for example Serum
ELISA) (to examine, for example, levels of protein expression),
biochemical enzymatic activity assays, in situ hybridization,
Northern analysis and/or PCR analysis of mRNAs, as well as any one
of the wide variety of assays that can be performed by gene and/or
tissue array analysis. Typical protocols for evaluating the status
of genes and gene products are found, for example in Ausubel et al.
eds., 1995, Current Protocols In Molecular Biology, Units 2
(Northern Blotting), 4 (Southern Blotting), 15 (immunoblotting) and
18 (PCR Analysis). A skilled artisan can readily adapt known
protein/antibody detection methods for use in determining whether
cells express a marker of the present invention and the relative
concentration of that specific polypeptide expression product in
blood or other body tissues.
[0082] In such alternative methods, a sample may be contacted with
an antibody specific for said biomarker under conditions sufficient
for an antibody-biomarker complex to form, and then detecting said
complex. The presence of the biomarker may be detected in a number
of ways, such as by Western blotting and ELISA procedures for
assaying a wide variety of tissues and samples, including plasma or
serum. A wide range of immunoassay techniques using such an assay
format are available, see, e.g., U.S. Pat. Nos. 4,016,043,
4,424,279 and 4,018,653. These include both single-site and
two-site or "sandwich" assays of the non-competitive types, as well
as in the traditional competitive binding assays. These assays also
include direct binding of a labeled antibody to a target
biomarker.
[0083] Sandwich assays are among the most useful and commonly used
assays. A number of variations of the sandwich assay technique
exist, and all are intended to be encompassed by the present
invention. Briefly, in a typical forward assay, an unlabelled
antibody is immobilized on a solid substrate, and the sample to be
tested brought into contact with the bound molecule. After a
suitable period of incubation, for a period of time sufficient to
allow formation of an antibody-antigen complex, a second antibody
specific to the antigen, labeled with a reporter molecule capable
of producing a detectable signal is then added and incubated,
allowing time sufficient for the formation of another complex of
antibody-antigen-labeled antibody. Any unreacted material is washed
away, and the presence of the antigen is determined by observation
of a signal produced by the reporter molecule. The results may
either be qualitative, by simple observation of the visible signal,
or may be quantitated by comparing with a control sample containing
known amounts of biomarker.
[0084] Variations on the forward assay include a simultaneous
assay, in which both sample and labeled antibody are added
simultaneously to the bound antibody. These techniques are well
known to those skilled in the art, including any minor variations
as will be readily apparent. In a typical forward sandwich assay, a
first antibody having specificity for the biomarker is either
covalently or passively bound to a solid surface. The solid surface
is typically glass or a polymer, the most commonly used polymers
being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene. The solid supports may be in the form of
tubes, beads, discs of microplates, or any other surface suitable
for conducting an immunoassay. The binding processes are well-known
in the art and generally consist of cross-linking covalently
binding or physically adsorbing, the polymer-antibody complex is
washed in preparation for the test sample. An aliquot of the sample
to be tested is then added to the solid phase complex and incubated
for a period of time sufficient (e.g. 2-40 minutes or overnight if
more convenient) and under suitable conditions (e.g. from room
temperature to 40.degree. C. such as between 25.degree. C. and
32.degree. C. inclusive) to allow binding of any subunit present in
the antibody. Following the incubation period, the antibody subunit
solid phase is washed and dried and incubated with a second
antibody specific for a portion of the biomarker. The second
antibody is linked to a reporter molecule which is used to indicate
the binding of the second antibody to the molecular marker.
[0085] An alternative method involves immobilizing the target
biomarkers in the sample and then exposing the immobilized target
to specific antibody which may or may not be labeled with a
reporter molecule. Depending on the amount of target and the
strength of the reporter molecule signal, a bound target may be
detectable by direct labeling with the antibody. Alternatively, a
second labeled antibody, specific to the first antibody is exposed
to the target-first antibody complex to form a target-first
antibody-second antibody tertiary complex. The complex is detected
by the signal emitted by the reporter molecule. By "reporter
molecule", as used in the present specification, is meant a
molecule which, by its chemical nature, provides an analytically
identifiable signal which allows the detection of antigen-bound
antibody. The most commonly used reporter molecules in this type of
assay are either enzymes, fluorophores or radionuclide containing
molecules (i.e. radioisotopes) and chemiluminescent molecules.
[0086] In the case of an enzyme immunoassay, an enzyme is
conjugated to the second antibody, generally by means of
glutaraldehyde or periodate. As will be readily recognized,
however, a wide variety of different conjugation techniques exist,
which are readily available to the skilled artisan. Commonly used
enzymes include horseradish peroxidase, glucose oxidase,
-galactosidase and alkaline phosphatase, amongst others. The
substrates to be used with the specific enzymes are generally
chosen for the production, upon hydrolysis by the corresponding
enzyme, of a detectable color change. Examples of suitable enzymes
include alkaline phosphatase and peroxidase. It is also possible to
employ fluorogenic substrates, which yield a fluorescent product
rather than the chromogenic substrates noted above. In all cases,
the enzyme-labeled antibody is added to the first
antibody-molecular marker complex, allowed to bind, and then the
excess reagent is washed away. A solution containing the
appropriate substrate is then added to the complex of
antibody-antigen-antibody. The substrate will react with the enzyme
linked to the second antibody, giving a qualitative visual signal
which may be further quantitated. Usually spectrophotometrically,
to give an indication of the amount of biomarker which was present
in the sample. Alternately, fluorescent compounds, such as
fluorescein and rhodamine, may be chemically coupled to antibodies
without altering their binding capacity. When activated by
illumination with light of a particular wavelength, the
fluorochrome-labeled antibody adsorbs the light energy, inducing a
state to excitability in the molecule, followed by emission of the
light at a characteristic color visually detectable with a light
microscope. As in the ETA, the fluorescent labeled antibody is
allowed to bind to the first antibody-molecular marker complex.
After washing off the unbound reagent, the remaining tertiary
complex is then exposed to the light of the appropriate wavelength,
the fluorescence observed indicates the presence of the molecular
marker of interest. Immunofluorescence and EIA techniques are both
very well established in the art. However, other reporter
molecules, such as radioisotope, chemiluminescent or bioluminescent
molecules, may also be employed.
[0087] Methods of the invention further include protocols which
examine the presence and/or expression of mRNAs, in a tissue or
cell sample. Methods for the evaluation of mRNAs in cells are well
known and include, for example, hybridization assays using
complementary DNA probes (such as in situ hybridization using
labeled riboprobes, Northern blot and related techniques) and
various nucleic acid amplification assays (such as RT-PCR and other
amplification type detection methods, such as, for example,
branched DNA, SISBA, TMA and the like).
[0088] In an embodiment, the level of mRNA corresponding to the
marker can be determined both by in situ and by in vitro formats in
a biological sample using methods known in the art. Many expression
detection methods use isolated RNA. For in vitro methods, any RNA
isolation technique that does not select against the isolation of
mRNA can be utilized for the purification of RNA from cells. See,
e.g., Asubel et al., Ed., Curr. Prot. Mol. Biol., John Wiley &
Sons, NY (1987-1999). Additionally, large numbers of tissue samples
can readily be processed using techniques well-known to those of
skill in the art, such as, e.g., the single-step RNA isolation
process of U.S. Pat. No. 4,843,155. The isolated mRNA can be used
in hybridization or amplification assays that include, but are not
limited to, Southern or Northern analyses, PCR analyses and probe
arrays. One preferred diagnostic method for the detection of mRNA
levels involves contacting the isolated mRNA with a nucleic acid
molecule (probe) that can hybridize to the mRNA encoded by the gene
being detected. The nucleic acid probe can be, e.g., a full-length
cDNA, or a portion thereof, such as an oligonucleotide of at least
7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient
to specifically hybridize under stringent conditions to a mRNA or
genomic DNA encoding a marker of the present invention. Other
suitable probes for use in the diagnostic assays of the invention
are described herein. Hybridization of an mRNA with the probe
indicates that the marker in question is being expressed.
[0089] In one format, the mRNA is immobilized on a solid surface
and contacted with a probe, for example, by running the isolated
mRNA on an agarose gel and transferring the mRNA from the gel to a
membrane, such as nitrocellulose. In an alternative format, the
probe(s) are immobilized on a solid surface and the mRNA is
contacted with the probe(s), for example, in an Affymetrix gene
chip array. A skilled artisan can readily adapt known mRNA
detection methods for use in detecting the level of mRNA encoded by
the markers of the present invention.
[0090] Although amplification of molecules is not required in the
present invention as discussed in the examples section, one of
skill in the art could use amplification methods. One alternative
method for determining the level of mRNA corresponding to a marker
of the present invention in a sample involves the process of
nucleic acid amplification, e.g., by RT-PCR (the experimental
embodiment set forth in Mullis, U.S. Pat. No. 4,683,202 (1987);
ligase chain reaction, self-sustained sequence replication,
Guatelli et al., Proc. Natl. Acad. Sci. USA, Vol. 87, pp. 1874-1878
(1990); transcriptional amplification system, Kwoh al, Proc. Natl.
Acad. Sci. USA, Vol. 86, pp, 1173-1177 (1989); Q-Beta Replicase,
Lizardi et al., Biol. Technology, Vol. 6, p. 1197 (1988); rolling
circle replication, U.S. Pat. No. 5,854,033 (1988); or any other
nucleic acid amplification method, followed by the detection of the
amplified molecules using techniques well-known to those of skill
in the art. These detection schemes are especially useful for the
detection of the nucleic acid molecules if such molecules are
present in very low numbers. As used herein, amplification primers
are defined as being a pair of nucleic acid molecules that can
anneal to 5' or 3' regions of a gene (plus and minus strands,
respectively, or vice-versa) and contain a short region in between.
In general, amplification primers are from about 10-30 nucleotides
in length and flank a region from about 50-200 nucleotides in
length. Under appropriate conditions and with appropriate reagents,
such primers permit the amplification of a nucleic acid molecule
comprising the nucleotide sequence flanked by the primers.
[0091] For in situ methods, mRNA does not need to be isolated form
the cells prior to detection. In such methods, a cell or tissue
sample is prepared/processed using known histological methods. The
sample is then immobilized on a support, typically a glass slide,
and then contacted with a probe that can hybridize to mRNA that
encodes the marker.
[0092] As an alternative to making determinations based on the
absolute expression level of the marker, determinations may be
based on the normalized expression level of the marker. Expression
levels are normalized by correcting the absolute expression level
of a marker by comparing its expression to the expression of a gene
that is not a marker, e.g., a housekeeping gene that is
constitutively expressed. Suitable genes for normalization include
housekeeping genes, such as the actin gene or epithelial
cell-specific genes. This normalization allows the comparison of
the expression level in one sample, e.g., a patient sample, to
another sample or between samples from different sources.
[0093] Alternatively, the expression level can be provided as a
relative expression level. To determine a relative expression level
of a marker, the level of expression of the marker is determined
for 10 or more samples of normal versus disease biological samples,
preferably 50 or more samples, prior to the determination of the
expression level for the sample in question. The mean expression
level of each of the genes assayed in the larger number of samples
is determined and this is used as a baseline expression level for
the marker. The expression level of the marker determined for the
test sample (absolute level of expression) is then divided by the
mean expression value obtained for that marker. This provides a
relative expression level.
[0094] Preferably, the samples used in the baseline determination
will be from patients who do not have the polymorphism. The choice
of the cell source is dependent on the use of the relative
expression level. Using expression found in normal tissues as a
mean expression score aids in validating whether the marker assayed
is specific (versus normal cells). In addition, as more data is
accumulated, the mean expression value can be revised, providing
improved relative expression values based on accumulated data.
Antibodies and Aptamers
[0095] In a preferred embodiment, the antibodies and aptamers
specifically bind each component of the biomarkers described
herein. The components include the nucleic acid sequences,
complementary sequences, fragments, alleles, variants and gene
products thereof of each component in each biomarker.
[0096] Aptamer polynucleotides are typically single-stranded
standard phosphodiester DNA (ssDNA). Close DNA analogs can also be
incorporated into the aptamer as described below.
[0097] A typical aptamer discovery procedure is described below: A
polynucleotide comprising a randomized sequence between "arms"
having constant sequence is synthesized. The arms can include
restriction sites for convenient cloning and can also function as
priming sites for PCR primers. The synthesis can easily be
performed on commercial instruments.
[0098] The target protein is treated with the randomized
polynucleotide. The target protein can be in solution and then the
complexes immobilized and separated from unbound nucleic acids by
use of an antibody affinity column. Alternatively, the target
protein might be immobilized before treatment with the randomized
polynucleotide.
[0099] The target protein-polynucleotide complexes are separated
from the uncomplexed material and then the bound polynucleotides
are separated from the target protein. The bound nucleic acid can
then be characterized, but is more commonly amplified, e.g. by PCR
and the binding, separation and amplification steps are repeated.
In many instances, use of conditions increasingly promoting
separation of the nucleic acid from the target protein, e.g. higher
salt concentration, in the binding buffer used in step 2) in
subsequent iterations, results in identification of polynucleotides
having increasingly high affinity for the target protein.
[0100] The nucleic acids showing high affinity for the target
proteins are isolated and characterized. This is typically
accomplished by cloning the nucleic acids using restriction sites
incorporated into the arms, and then sequencing the cloned nucleic
acid.
[0101] The affinity of aptamers for their target proteins is
typically in the nanomolar range, but can be as low as the
picomolar range. That is K.sub.D is typically 1 pM to 500 nM, more
typically from 1 pM to 100 nM. Aptamers having an affinity of
K.sub.D in the range of 1 pM to 10 nM are also useful.
[0102] Aptamer polynucleotides can be synthesized on a commercially
available nucleic acid synthesizer by methods known in the art. The
product can be purified by size selection or chromatographic
methods.
[0103] Aptamer polynucleotides are typically from about 10 to 200
nucleotides long, more typically from about 10 to 100 nucleotides
long, still more typically from about 10 to 50 nucleotides long and
yet more typically from about 10 to 25 nucleotides long. A
preferred range of length is from about 10 to 50 nucleotides.
[0104] The aptamer sequences can be chosen as a desired sequence,
or random or partially random populations of sequences can be made
and then selected for specific binding to a desired target protein
by assay in vitro. Any of the typical nucleic acid-protein binding
assays known in the art can be used, e.g. "Southwestern" blotting
using either labeled oligonucleotide or labeled protein as the
probe. See also U.S. Pat. No. 5,445,935 for a fluorescence
polarization assay of protein-nucleic acid interaction.
[0105] Appropriate nucleotides for aptamer synthesis and their use,
and reagents for covalent linkage of proteins to nucleic acids and
their use, are considered known in the art. A desired
aptamer-protein complex, for example, aptamer-thrombin complex of
the invention can be labeled and used as a diagnostic agent in
vitro in much the same manner as any specific protein-binding
agent, e.g. a monoclonal antibody. Thus, an aptamer-protein complex
of the invention can be used to detect and quantitate the amount of
its target protein in a sample, e.g. a blood sample, to provide
diagnosis of a disease state correlated with the amount of the
protein in the sample.
[0106] A desired aptamer-target/bait molecular complex can also be
used for diagnostic imaging, in imaging uses, the complexes are
labeled no that they can be detected outside the body. Typical
labels are radioisotopes, usually ones with short half-lives. The
usual imaging radioisotopes, such as .sup.123I, .sup.124I,
.sup.125I, .sup.131I, .sup.99mTC, .sup.186Re, .sup.188Re,
.sup.64Cu, .sup.67Cu, .sup.212Bi, .sup.213Bi, .sup.67Ga, .sup.90Y,
.sup.111In, .sup.18F, .sup.3H, .sup.35S or .sup.32P can be used.
Nuclear magnetic resonance (NMR) imaging enhancers, such as
gadolinium-153, can also be used to label the complex for detection
by NMR. Methods and reagents for performing the labeling, either in
the polynucleotide or in the protein moiety, are considered known
in the art.
[0107] In a preferred embodiment, an antibody or aptamer is
specific for each biomolecule of in Tables 1 to 19.
Drug Discovery
[0108] In other preferred embodiments, the molecular signatures are
useful for the identification of new drugs in the treatment of
cardiovascular diseases and disorders.
[0109] In another preferred embodiment, the molecular signatures
would verify whether a patient's treatment is progressing. For
example, the molecular signature may change during the course of
treatment and reflect normal controls.
[0110] Small Molecules: Small molecule test compounds or candidate
therapeutic compounds can initially be members of an organic or
inorganic chemical library. As used herein, "small molecules"
refers to small organic or inorganic molecules of molecular weight
below about 3,000 Daltons. The small molecules can be natural
products or members of a combinatorial chemistry library. A set of
diverse molecules should be used to cover a variety of functions
such as charge, aromaticity, hydrogen bonding, flexibility, size,
length of side chain, hydrophobicity, and rigidity. Combinatorial
techniques suitable for synthesizing small molecules are known in
the art, e.g., as exemplified by Obrecht and Villalgordo,
Solid-Supported Combinatorial and Parallel Synthesis of
Small-Molecular-Weight Compound Libraries, Pergamon-Elsevier
Science Limited (1998), and include those such as the "split and
pool" or "parallel" synthesis techniques, solid-phase and
solution-phase techniques, and encoding techniques (see, for
example, Czarnik, Curr. Opin. Chem. Bio., 1:60(1997). In addition,
a number of small molecule libraries are commercially
available.
[0111] Particular screening applications of this invention relate
to the testing of pharmaceutical compounds in drug research. The
reader is referred generally to the standard textbook "In vitro
Methods in Pharmaceutical Research", Academic Press, 1997, and U.S.
Pat. No. 5,030,015). Assessment of the activity of candidate
pharmaceutical compounds generally involves administering a
candidate compound, determining any change in the morphology,
marker phenotype and expression, or metabolic activity of the cells
and function of the cells that is attributable to the compound
(compared with untreated cells or cells treated with an inert
compound), and then correlating the effect of the compound with the
observed change.
[0112] The screening may be done, for example, either because the
compound is designed to have a pharmacological effect on certain
cell types, or because a compound designed to have effects
elsewhere may have unintended side effects. Two or more drugs can
be tested in combination (by combining with the cells either
simultaneously or sequentially), to detect possible drug-drug
interaction effects. In some applications, compounds are screened
initially for potential toxicity (Castell et al., pp, 375-410 in
"In vitro Methods in Pharmaceutical Research." Academic Press,
1997), Cytotoxicity can be determined in the first instance by the
effect on cell viability, survival, morphology, and expression or
release of certain markers, receptors or enzymes. Effects of a drug
on chromosomal DNA can be determined by measuring DNA synthesis or
repair. [.sup.3H]thymidine or BrdU incorporation, especially at
unscheduled times in the cell cycle, or above the level required
for cell replication, is consistent with a drug effect. Unwanted
effects can also include unusual rates of sister chromatid
exchange, determined by metaphase spread. The reader is referred to
A. Vickers (PP 375-410 in "In vitro Methods in Pharmaceutical
Research," Academic Press, 1997) for further elaboration.
[0113] In one embodiment of the invention, a method of identifying
a candidate agent is provided said method comprising: (a)
contacting a biological sample from a patient with the candidate
agent and determining the level of expression of one or more
biomarkers described herein; (b) determining the level of
expression of a corresponding biomarker or biomarkers in an aliquot
of the biological sample not contacted with the candidate agent;
(c) observing the effect of the candidate agent by comparing the
level of expression of the biomarker or biomarkers in the aliquot
of the biological sample contacted with the candidate agent and the
level of expression of the corresponding biomarker or biomarkers in
the aliquot of the biological sample not contacted with the
candidate agent; and (d) identifying said agent from said observed
effect, wherein an at least 1%, 2%, 5%, 10% difference between the
level of expression of the biomarker gene or combination of
biomarker genes in the aliquot of the biological sample contacted
with the candidate agent and the level of expression of the
corresponding biomarker gene, or combination of biomarker genes in
the aliquot of the biological sample not contacted with the
candidate agent is an indication of an effect of the candidate
agent.
[0114] In preferred embodiments, the effects of the drug are
correlated with the expression of the molecular signatures
associated with a good prognosis as described in detail in the
examples which follow.
[0115] In another embodiment of the invention, a candidate agent
derived by the method according to the invention is provided.
[0116] In another embodiment of the invention, a pharmaceutical
preparation comprising an agent according to the invention is
provided.
[0117] In another preferred embodiment of the invention, a method
of producing a drug comprising; the steps of the method according
to the invention (i) synthesizing the candidate agent identified in
step (c) above or an analog or derivative thereof in an amount
sufficient to provide said drug in a therapeutically effective
amount to a subject; and/or (ii) combining the drug candidate the
candidate agent identified in step (c) above or an analog or
derivative thereof with a pharmaceutically acceptable carrier.
[0118] Vectors, Cells: In some embodiments it is desirable to
express the biomolecules that comprise a biomarker, in a vector and
in cells. The applications of such combinations are unlimited. The
vectors and cells expressing the one or more biomolecules can be
used in assays, kits, drug discovery, diagnostics, prognostics and
the like. The cells can be stem cells isolated from the bone marrow
as a progenitor cell, or cells obtained from any other source, such
as for example, ATCC.
[0119] "Bone marrow derived progenitor cell" (BMDC) or "bone marrow
derived stem cell" refers to a primitive stem cell with the
machinery for self-renewal constitutively active. Included in this
definition are stem cells that are totipotent, pluripotent and
precursors. A "precursor cell" can be any cell in a cell
differentiation pathway that is capable of differentiating into a
more mature cell. As such, the term "precursor cell population"
refers to a group of cells capable of developing into amore mature
cell. A precursor cell population can comprise cells that are
totipotent, cells that are pluripotent and cells that are stem cell
lineage restricted (i.e. cells capable of developing into less than
all hematopoietic lineages, or into, for example, only cells of
erythroid lineage). As used herein, the term "totipotent cell"
refers to a cell capable of developing into all lineages of cells.
Similarly, the term "totipotent population of cells" refers to a
composition of cells capable of developing into all lineages of
cells. Also as used herein, the term "pluripotent cell" refers to a
cell capable of developing into a variety (albeit not all) lineages
and are at least able to develop into all hematopoietic lineages
(e.g., lymphoid, erythroid, and thrombocytic lineages). Bone marrow
derived stem cells contain two well-characterized types of stem
cells. Mesenchymal stem cells (MSC) normally form chondrocytes and
osteoblasts. Hematopoietic stem cells (HSC) are of mesodermal
origin that normally gives rise to cells of the blood and immune
system (e.g., erythroid, granulocyte/macrophage, magakaryocite and
lymphoid lineages). In addition, hematopoietic stem cells also have
been shown to have the potential to differentiate into the cells of
the liver (including hepatocytes, bile duct cells), lung, kidney
(e.g., renal tubular epithelial cells and renal parenchyma),
gastrointestinal tract, skeletal muscle fibers, astrocytes of the
CNS. Purkinje neurons, cardiac muscle (e.g., cardiomyocytes),
endothelium and skin.
[0120] In a preferred embodiment, a method of identifying candidate
therapeutic compounds comprises culturing cells expressing at least
one biomolecule selected from biomarker signatures in Tables 1 to
19.
[0121] Such compounds are useful, e.g., as candidate therapeutic
compounds for the treatment of heart disease, heart disorders and
conditions thereof. Thus, included herein are methods for screening
for candidate therapeutic compounds for the treatment of, for
example, myocarditis, Coronary Heart Disease, angina, Acute
Coronary Syndrome, Aortic Aneurysm and Dissection, arrhythmias,
Cardiomyopathy, Congenital Heart Disease, congestive heart failure
or chronic heart failure, pericarditis, and the like. The methods
include administering the compound to a model of the condition,
e.g., contacting a cell (in vitro) model with the compound, or
administering the compound to an animal model of the condition,
e.g., an animal model of a condition associated with heart disease.
The model is then evaluated for an effect of the candidate compound
on the clinical outcome in the model and can be considered a
candidate therapeutic compound for the treatment of the condition.
Such effects can include clinically relevant effects, decreased
pain; increased life span; and so on. Such effects can be
determined on a macroscopic or microscopic scale, Candidate
therapeutic compounds identified by these methods can be further
verified, e.g., by administration to human subjects in a clinical
trial.
[0122] The biomolecules can be expressed from one or more vectors.
A "vector" (sometimes referred to as gene delivery or gene transfer
"vehicle") refers to a macromolecule or complex of molecules
comprising a polynucleotide to be delivered to a host cell, either
in vitro or in vivo. The polynucleotide to be delivered may
comprise a coding sequence of interest in gene therapy. Vectors
include, for example, viral vectors (such as adenoviruses ("Ad"),
adeno-associated viruses (AAV), and retroviruses), liposomes and
other lipid-containing complexes, and other macromolecular
complexes capable of mediating delivery of a polynucleotide to a
host cell. Vectors can also comprise other components or
functionalities that further modulate gene delivery and/or gene
expression, or that otherwise provide beneficial properties to the
targeted cells. As described and illustrated in more detail below,
such other components include, for example, components that
influence binding or targeting to cells (including components that
mediate cell-type or tissue-specific binding); components that
influence uptake of the vector nucleic acid by the cell; components
that influence localization of the polynucleotide within the cell
after uptake (such as agents mediating nuclear localization); and
components that influence expression of the polynucleotide. Such
components also might include markers, such as detectable and/or
selectable markers that can be used to detect or select for cells
that have taken up and are expressing the nucleic acid delivered by
the vector. Such components can be provided as a natural feature of
the vector (such as the use of certain viral vectors which have
components or functionalities mediating binding and uptake), or
vectors can be modified to provide such functionalities. Other
vectors include those described by Chen et al; BioTechniques, 34:
167-171 (2003). Large varieties of such vectors are known in the
art and are generally available.
[0123] In another preferred embodiment, a vector expresses one or
more biomolecules identified in any one or more of Tables 1 to
19.
Kits
[0124] In another preferred embodiment, a kit is provided
comprising any one or more of the biomarkers or molecular
signatures comprising Tables 1 to 19.
[0125] For use in the applications described or suggested above,
kits or articles of manufacture are also provided by the invention.
Such kits may comprise a carrier means being compartmentalized to
receive in close confinement one or more container means such as
vials, tubes, and the like, each of the container means comprising
one of the separate elements to be used in the method. For example,
one of the container means may comprise a probe that is or can be
detectably labeled. Where the kit utilizes nucleic acid
hybridization to detect the target nucleic acid, the kit may also
have containers containing nucleotide(s) for amplification of the
target nucleic acid sequence and/or a container comprising a
reporter-means, such as a biotin-binding protein, such as avidin or
streptavidin, bound to a reporter molecule, such as an enzymatic,
florescent, or radioisotope label.
[0126] The kit of the invention will typically comprise the
container described above and one or more other containers
comprising materials desirable from a commercial and user
standpoint, including buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use. A label
may be present on the container to indicate that the composition is
used for a specific therapy or non-therapeutic application, and may
also indicate directions for either in vivo or in vitro use, such
as those described above.
[0127] The kits of the invention have a number of embodiments. A
typical embodiment is a kit comprising a container, a label on said
container, and a composition contained within said container;
wherein the composition includes a primary antibody that binds to
the biomolecules of each molecular signature and instructions for
using the antibody for evaluating the presence of biomolecules in
at least one type of mammalian cell The kit can further comprise a
set of instructions and materials for preparing a tissue sample and
applying antibody and probe to the same section of a tissue sample.
The kit may include both a primary and secondary antibody, wherein
the secondary antibody is conjugated to a label, e.g., an enzymatic
label.
[0128] Another embodiment is a kit comprising a container, a label
on said container, and a composition contained within said
container; wherein the composition includes a polynucleotide that
hybridizes to a complement of the polynucleotides under stringent
conditions, the label on said container indicates that the
composition can be used to evaluate the presence of a molecular
signature in at least one type of mammalian cell, and instructions
for using the polynucleotide for evaluating the presence of
biomolecule RNA or DNA in at least one type of mammalian cell.
[0129] Other optional components in the kit include, microarrays,
one or more buffers (e.g., block buffer, wash buffer, substrate
buffer, etc), other reagents such as substrate (e.g., chromogen)
which is chemically altered by an enzymatic label, epitope
retrieval solution, control samples (positive and/or negative
controls), control slide(s) etc.
[0130] The invention has been described in detail with reference to
preferred embodiments thereof. However, it will be appreciated that
those skilled in the art, upon consideration of this disclosure,
may make modifications and improvements within the spirit and scope
of the invention. The following non-limiting examples are
illustrative of the invention.
[0131] All documents mentioned herein are incorporated herein by
reference. All publications and patent documents cited in this
application are incorporated by reference for all purposes to the
same extent as if each individual publication or patent document
were so individually denoted. By their citation of various
references in this document, Applicants do not admit any particular
reference is "prior art" to their invention.
EXAMPLES
[0132] The following non-limiting Examples serve to illustrate
selected embodiments of the invention. It will be appreciated that
variations in proportions and alternatives in elements of the
components shown will be apparent to those skilled in the art and
are within the scope of embodiments of the present invention.
[0133] Embodiments of the invention may be practiced without the
theoretical aspects presented. Moreover, the theoretical aspects
are presented with the understanding that Applicants do not seek to
be bound by the theory presented.
[0134] While various embodiments of the present invention have been
described above, it should be understood that they have been
presented by way of example only, and not limitation. Numerous
changes to the disclosed embodiments can be made in accordance with
the disclosure herein without departing from the spirit or scope of
the invention. Thus, the breadth and scope of the present invention
should not be limited by any of the above described
embodiments.
Materials and Methods:
[0135] Clinical evaluation of patients: Transcriptomic analysis of
heart tissue was performed in matched cohorts of patients with IDCM
(n=32) and myocarditis (n=16) selected from a biorepository
containing samples from patients with new onset heart failure (HF;
n=350). Patients underwent EMB as part of a comprehensive
diagnostic evaluation of heart dysfunction that included history
and physical exam, right-heart cardiac catheterization and
echocardiography. All patients with history suggestive for ischemic
heart disease or at least two standard risk factors for
atherosclerosis were further evaluated with coronary angiography.
Blood tests were performed for cardiac enzymes, thyroid-function
and antinuclear antibodies.
[0136] Four to six biopsy specimens were obtained from each patient
and examined by an experienced cardiac pathologist. In addition to
standard staining, Congo red was used to identify amyloidosis and
Prussian blue if hemochromatosis was suspected. Myocarditis was
defined according to Dallas criteria, without additional tests for
presence of viral RNA, such as PCR.
[0137] After this extensive evaluation, idiopathic dilated
cardiomyopathy (IDCM) was a diagnosis of exclusion. In addition to
diagnostic biopsies, one sample was flash frozen and stored in
liquid nitrogen for microarray analysis. All participants gave
written informed consent for collection of samples and clinical
data. Right ventricular septal EMBs were obtained by advancing a
disposable bioptome (Argon; Jawz via the right jugular vein under
fluoroscopic guidance.
[0138] Selection of patients: A total of 75 samples were used for
microarray analysis. Forty-eight samples were selected for the
first transcriptomic study. These included samples from patients
with myocarditis (n=16) defined by the Dallas criteria and
idiopathic dilated cardiomyopathy (IDCM, n=32) selected in a
case-control fashion based on age, gender, functional parameters
from echocardiography and right heart catheterization, and
medication usage. In addition, samples from 6 patients were
identified with myocarditis with divergent baseline criteria, from
which the diagnostic accuracy of the biomarker was independently
tested. Finally, RNA was prepared from samples obtained from
patients with rare but clinically significant variants of
inflammatory heart disease--cardiac sarcoidosis (n=9), giant
myocarditis (n=3), peripartum cardiomyopathy (n=6), and heart
failure in the setting of systemic lupus erythematosus (n=3).
[0139] RNA extraction and microarray hybridization: Total RNA was
extracted from biopsies as previously described. Quality control of
integrity of RNA was performed with the 2100 Bioanalyzer (Agilent).
MIAME guidelines were followed for all steps of the procedure. The
extracted RNA (average 568.+-.88 ng; Standard Error of the Mean
(SEM)) was preprocessed with the Ovation Biotin RNA Amplification
and Labeling System (NuGen, Cat. No. 2300-12) for subsequent
hybridization with the Human Genome U133 Plus 2.0 Array from
Affymetrix without additional amplification step.
[0140] Bioinformatic and biostatistic software: Microarray data was
normalized with Robust Multiarray Average (RMA) and analyzed with
Significance Analysis of Microarrays (SAM) to identify
differentially expressed genes in patients with myocarditis (n=16)
vs IDCM (n=32). The resulting gene list was further processed with
Meta Core pathway analysis incorporated in GeneGo (bioinformatics
software, St. Joseph, Mich.). Organ- and species-specific
pre-filtering was performed before network analysis, in order to
extract solely pathways that are truly interrelated in the human
heart. Each network was provided with a p-value, using the basic
formula for hypergeometric distribution. This formula provides a
value that represents the probability for a particular mapping of
an experiment to a map (or network/process) to arise by chance,
considering the numbers of genes in the experiment vs the number of
genes in the map within the "full set" of all genes on maps.
[0141] In addition, a z-score was calculated for each network,
which reflects the saturation with genes from the experiment. A
high z-score indicates a network that contains a large amount of
genes from the experiment.
[0142] In order to determine the minimum number of differentially
expressed genes required for detection of patients with myocarditis
compared to IDCM, Prediction Analysis of Microarrays (PAM) was used
to obtain a biomarker based upon a nearest shrunken centroid. The
classifier was developed from a train set (n=33), consisting of 2/3
of data, and applied to an independent test set (n=15) containing
1/3 of data.
[0143] After developing the transcriptomic biomarker with a
case-control design, its performance was tested in unmatched
samples, to test its generalizability independent of age, gender,
heart function or drug therapy. To test this hypothesis, samples
from patients with myocarditis (n=6) were used, who presented with
higher ejection fractions (65.+-.4.7%). Finally, the molecular
signature was illustrated as a heatmap by an unsupervised
hierarchical clustering approach in R based on Euclidean
distance.
[0144] Then PAM was used to identify molecular signatures in
samples from patients with giant cell myocarditis (n=3),
sarcoidosis (n=9), peripartum cardiomyopathy (n=6) and systemic
lupus erythematosus (n=3), which distinguish them both from IDCM as
well as myocarditis and further refine diagnosis between
sarcoidosis and giant cell myocarditis.
[0145] In order to test, if previously established classification
algorithms can further reduce the number of genes necessary for
accurate prediction, misclassification-penalized posteriors
classification (MiPP) were applied, which successfully predicts
rejection in liver transplant recipients. The MiPP package is an
application in the R environment, which employs the libraries MASS
for lda/qda (linear/quadratic discriminant analysis and e1071 for
SVM (support vector machine). This software sequentially adds genes
to a classification model based upon the Misclassication-Penalized
Posteriors principle, which takes into account the likelihood that
a sample belongs to a given class by using posterior probability of
correct classification.
[0146] First MiPP was used to test several different classification
rules, to further reduce the novel molecular signature, consisting
of 62 genes. Support vector machine was subsequently applied with
radial basis function (SVM-rbf) and lineal function (SVM-lin),
quadratic discriminant analysis (qda), linear discriminant analysis
(lda) and a combination of lda, qda and svm-rbf. When support
vector machine algorithms are used for classification, the input
data is plotted as two vectors in an n-dimensional space and a
virtual hyperplane is created that best separates the two
phenotypes. This hyperplane is then used to classify samples with
unknown phenotypes. Linear discriminant analysis uses a linear
combination of features, which best separate two or more classes.
Quadratic discriminant analysis is closely related to lda, however
there is no assumption that the covariance of each of the classes
is identical. Models were developed based upon 5-fold cross
validation in a train set (2/3 of data) and subsequent validation
in an independent test set (1/3 of data).
[0147] In order to evaluate, if distinct models are generated from
additional random splits, 50 random divisions were performed to
develop individual classification models, which were then validated
in 200 independent splits. As an additional confirmatory test,
principal components analysis (PCA) was performed to illustrate how
well patients with myocarditis can be separated from patients with
IDCM based on the original 62 genes molecular signature, and to
test if genes that were identified by MiPP analysis to be the most
robust classifiers, would also be discovered to be important when
PCA was applied. PCA is a method that depicts the importance of
genes for phenotypic classification by means of illustration
through Eigen vectors towards a phenotype, in which the gene is
overexpressed. If genes are less robust as classifiers, the
corresponding vector directs towards the center with close to
vertical direction. Important classifiers are depicted with vectors
having endpoints far from the center.
[0148] Further testing of the diagnostic biomarker for myocarditis
in a previously published data set: In order to test, if the
developed transcriptomic diagnostic biomarker enables detection of
myocarditis entirely independent samples, that were collected and
processed at a different time point, a previously published dataset
derived from patients with either giant cell myocarditis (n=3) or
normal heart (n=11) and processed with the previous U133A
microarray (Affymetrix) was used.
[0149] Validation of microarrays with quantitative realtime RT-PCR:
Validation with realtime RT-PCR was performed in a randomly
selected subset of patients (IDCM: n=10, myocarditis: n=10), with
triplicates replication. First-strand cDNA was synthesized with a
High-Capacity cDNA Reverse-Transcription Kit (Applied Biosystems
Inc., CA, USA) from 100 ng total RNA, which was amplified with
MessageAmp II Amplification Kit (Applied Biosystems Inc., CA, USA).
TaqMan probes, labeled with 6-carboxyfluorescein (FAM) were
designed for a subset of differentially expressed genes identified
by microarray analysis: CD14, FCER1G, TLR1, TLR2, TLR7, SIGLEC 1,
ADCY7, MEGF9, PTPLAD1, SWAP70, MSI1, LCE1E and HLA-DQ1, as well as
the housekeeping gene 18S RNA. Data were analyzed by the threshold
cycle (Ct) relative-quantification method (error bars=mean standard
error).
Example 1
Diagnostic Transcriptomic Biomarkers in Inflammatory
Cardiomyopathies
[0150] Table 13 depicts the baseline clinical variables of patients
included in the initial case-control population with idiopathic
dilated cardiomyopathy (IDCM) and Dallas criteria defined
lymphocytic myocarditis. By design, there were no differences in
gender, age, functional parameters or medication between the two
groups.
[0151] Discovery of phenotype specific differences in gene
expression and involved pathways: To identify differential gene
expression between patients with IDCM (n=32) and those with
lymphocytic myocarditis (n=1.6), oligonucleotide microarrays were
used to analyze RNA obtained from endomyocardial biopsies (EMBs)
from affected patients at first presentation with new onset heart
failure. 9,878 differentially expressed genes (q<5%, fold change
(FC)>1.2) were identified in patients with IDCM compared to
myocarditis (FIG. 1). Transcripts with FC>2 (141 over-expressed
and 16 down-regulated transcripts) are provided as in Tables 13 and
14. Pathway analysis with GeneGo Metacore revealed overexpression
of a total of 8 networks in myocarditis vs IDCM (Table 3). No
specific networks were revealed within the small amount of
down-regulated transcripts with FC>2 (16 genes).
[0152] Identification of a molecular signature to distinguish
myocarditis from non-inflammatory cardiomyopathy patients:
Prediction analysis of microarrays (PAM) were applied in a training
set containing 2/3 of data (IDCM: n=22; myocarditis: n=11) and
evaluated its accuracy in an independent test set, containing 1/3
of data (IDCM: n=10; myocarditis: n=5). The developed
transcriptomic diagnostic biomarker consisted of a minimal set of
62 transcripts (Table 14). When the molecular signature was tested
in the matched independent samples (n=15), it performed with 100%
accuracy (sensitivity: 100%, 95 CI: 46-100%; specificity: 100%, 95
CI: 66-100%; positive predictive value, PPV: 100%, 95 CI: 46-100%;
negative predictive value, NPV: 100%, 95 CI: 66-100%; FIG. 2). All
samples were predicted correctly, independent of the degree of
inflammation--borderline or active myocarditis.
[0153] Next, the transcriptomic diagnostic biomarker was tested in
an additional set of independent samples derived from patients with
myocarditis (n=6), who presented with higher ejection fractions
(65.+-.4.7%), compared to the case-control samples. In this group,
the molecular signature still had a high degree of diagnostic
accuracy and identified 83% of patients with myocarditis correctly
(sensitivity: 91%, 95 CI: 57-100%; specificity: 100%, 95 CI:
66-100%; PPV: 100%, 95 CI: 66-100%; NPV: 91%, 95 CI: 57-100%).
[0154] Additional identification of gene models with recently
established classification strategies: In order to obtain a more
parsimonious molecular signature several bioinformatic approaches
were employed, followed by quantitative realtime RT-PCR validation.
First, multiple established classification algorithms were applied
using the MiPP package in R that includes lineal discriminant
analysis (lda), quadratic discriminant analysis (qda), supervector
machine with radial basis function (svm-rbf), and supervector
machine with lineal function as kernel (svm-lin). When applied to
the 62 gene signature, these algorithms revealed that a 4 gene
subset signature would be diagnostic. Table 15 contains the mean
error for each established set of genes developed by individual
rules or combination of rules. Using these algorithms, a highly
diagnostic set of four genes (mean error of 0.167 in independent
validation sets (n=18)).
[0155] Since this was a random split into train and test set, this
analysis was continued by testing if a different random split of
data would reveal distinct models. Splitting of data into train
(2/3) and test set (1/3) and selecting a model for a given split
were repeated 50 times. For each split, the parsimonious model
identified was further evaluated by 200 independent splits. KRT78,
MSI1, POU4F1, LCE1 and the EST 1556507_at were selected as top
classifiers, with a mean error of 0.086 after validation in 200
independent splits (table 16). Mean sMiPP is an additional measure
for performance of a given gene model, approximating 1 with
increasing accuracy. When the top 5 gene models (Table 16) were
validated in 200 independent random splits, a mean sMiPP was
obtained ranging from 0.776-0.791 (Table 16). Since those models
were built from 50 initial random splits, it is likely that
identical gene clusters are identified in subsequent splits, as it
occurred in this analysis (Table 16: split #17 and split #45).
[0156] Validation of significance of genes for phenotypic
characterization by principal components analysis (PCA): PCA is a
valuable tool to illustrate importance of individual genes for
classification of their corresponding phenotype. In agreement with
results from the MiPP analysis, the transcripts 1556507_at, KRT78,
LCE1E, MSI1 and POU4F1 were identified as highly important, with
vectors having their endpoints distant from the center (FIG. 5A).
Additional genes that were revealed to be highly robust were ITGB2,
HERC6, ADCY7, NEK3, MEGF9, as well as the ESTs 1558605_at and
1565662_at. In addition, PCA clustered patients with similar
expression patterns as one principal component (PC). As visible in
FIG. 4B, samples from patients with myocarditis noticeably
separated from patients with 1DCM.
[0157] Validation of transcriptomic data with quantitative realtime
RT-PCR: To obtain technical validation of the results from
microarray analysis, realtime RT-PCR was performed on a subset of
16 genes (Table 17). Genes were selected from the resulting gene
lists of the bioinformatic approach, based on biological
plausibility and robustness as classifiers for lymphocytic
myocarditis.
[0158] This approach confirmed overrepresentation of HLA-DQ1+
patients in myocarditis (60%), while only 20% of patients with IDCM
were positive for DQ1. Fold change (FC) of most genes measured by
quantitative realtime RT-PCR strongly correlated with data obtained
from microarray analysis, except for MSI1, where realtime RT-PCR
data revealed much stronger downregulation in patients with
myocarditis vs lymphocytic cardiomyopathy than obtained from the
microarray data. Genes that were revealed by realtime RT-PCR to
have highest fold changes were CD14 (FC=+6.8), FCER1G (FC=+5), TLR1
(FC=+4.2), TLR2 (FC=+5.9), SIGLEC1 (FC=+4.3) and ADCY7 (+4.2)
(Table 17). However, among the 4 genes that were revealed by MiPP
analysis, KRT78 and POU4F1 could not be confirmed with realtime
RT-PCR. Since KRT78 appeared highly robust as classifier based on
the microarray results, two different primer pairs were used to
detect either the 3' or the 5' end of the gene sequence. However,
none of them were able to detect KRT78 in any of the samples. When
total RNA was used from immortalized keratinocytes as a positive
control, a signal was received from each primer pair. In order to
exclude the possibility of cross-hybridization that may have
occurred on the microarray assay, a batch search in the NCBI,
database (blast.ncbi.nlm.nih.gov/Blast.cgi) of the target sequence
that was used on the Affymetrix chip. However, there was no
significant sequence homology with any gene other than KRT78.
Despite this minimal incoherence between microarray analysis and
the more specific realtime RT-PCR, the diagnostic biomarker was
minimized to a very small set of 13 genes that performed highly
robust with both methods (100% sensitivity, 100% specificity). When
applied to a subset of myocarditis patients with higher ejection
fraction, the 13 gene signature performed with a sensitivity of 75%
(95CI: 36-96%), specificity of 100% (95CI: 52-100%), PPV of 100%
(95CI: 52-100%) and NPV of 75% (95CI: 36-96%).
[0159] Subtyping of inflammatory cardiomyopathies with diagnostic
transcriptomic biomarkers: It was then sought to test if rare
subtypes of inflammatory cardiomyopathy can be distinguished from
IDCM using TBBs. Molecular signatures containing 8 to 56 genes were
identified that identified patients with (a) giant cell myocarditis
(n=3), (b) sarcoidosis (n=9) and (c) peripartum cardiomyopathy
(n=9) with very high accuracy (up to 86%, Table 18). Further it was
sought to test the possibility of refining the diagnosis within the
group of inflammatory cardiomyopathies and to distinguish these
rare disorders from the more common lymphocytic myocarditis. While
patients with giant cell myocarditis and sarcoidosis each contained
a very robust cluster of genes, with an overall accuracy of 92% and
94% respectively, peripartum cardiomyopathy appeared to be less
distinct in its transcriptome with a molecular signature that
performed only with 69% overall accuracy (Table 19), likely
reflecting a spectrum of etiologies of this condition. Gene lists
of each classifier are provided in Tables 5-11.
[0160] After obtaining these compelling results, it was sought to
evaluate, if diagnosis between sarcoidosis and giant cell
myocarditis, two subtypes of inflammatory cardiomyopathy that
strongly resemble each other by histology, could be further
refined. A molecular signature was developed that identified
patients with giant cell myocarditis vs sarcoidosis based on a
classifier of 19 genes with 67% sensitivity (95 CI: 13-98%), 75%
specificity (95 CI: 36-96%), PPV of 50% (95 CI: 9-91%) and NPV of
86% (95 CI: 43-99%; Table 12).
[0161] Discussion:
[0162] Distinction of inflammatory as compared to non-inflammatory
cardiomyopathies by standard histology has, prior to this study,
represented a major diagnostic challenge. Moreover, delineating
between different inflammatory cardiomyopathies with highly
variable clinical courses has been, prior to this study, an even
more challenging task. Given the emerging value of transcriptomics
to add greatly to the accuracy of complex diagnoses, this approach
was applied to the problem of diagnostic inaccuracy in inflammatory
diseases of the heart, and here in, report the success with this
approach.
[0163] Inflammatory disorders of the heart have been, prior to this
study, notoriously difficult to diagnose due to the patchy nature
of the inflammation. In addition, a wide variety of underlying
inflammatory conditions, with highly variable clinical outcomes,
can affect the heart. Here the transcriptome obtained from a single
endomyocardial biopsy was employed to develop biomarkers that
enhanced the diagnostic accuracy for detection of cardiac
inflammation as well at the ability to separate between important
subtypes of cardiac inflammation. This approach illustrated the
value of the transcriptome as a diagnostic biomarker for heart
diseases and offers insights into anew clinically useful tool. The
data herein evidence the results obtained using the TBBs to
distinguish between idiopathic and ischemic cardiomyopathy and to
predict long term prognosis in new onset dilated
cardiomyopathy.
[0164] The discoveries reported here are clinically relevant as
high diagnostic sensitivity cardiomyopathy facilitates the
appropriate use of new myocarditis specific therapies. Early and
accurate diagnosis in this condition is essential so as to avoid
excessive myocardial damage resulting from failure to apply
therapies. New candidate therapies for myocyarditis include
anti-inflammatory cytokines, anti-viral agents, and
immunoabsorption. In this regard, IFN therapy has been safely
applied in humans, leading to increased LV function and elimination
of viral infection. Immunoglobin administration in acute
myocarditis as well as application of Ca-channel blockers, are
potential approaches with promising preliminary data that entail
further evaluation. While the use of immunosuppression in
inflammatory cardiomyopathy is highly controversial, there is
growing consensus that the identification of the relevant subtype
of inflammatory cardiomyopathy is crucial for successful treatment.
Accurate diagnosis is also critical for prognostic assessment,
since clinical outcome in inflammatory cardiomyopathies correlates
with disease etiology. TBBs add valuable information to a
comprehensive diagnostic evaluation of new onset heart failure.
[0165] In order to achieve an accurate biomarker a broad range of
bioinformatic approaches were employed. These included SAM, PAM,
MiPP, unsupervised hierarchical clustering and PCA. Using SAM, a
large number of differentially expressed genes in patients with
lymphocytic myocarditis vs idiopathic dilated cardiomyopathy were
identified. Importantly, differentially expressed genes involved
multiple biological networks with inflammatory components. Using
these differentially expressed genes, a subset were indentified
that functioned as a highly accurate biomarker, performing with
perfect accuracy, using nearest shrunken centroids.
[0166] To find the smallest set of genes for classification,
SVM-rbf, SVM-lin, QDA, LDA and a combination of LDA, QDA and
SVM-rbf in MiPP were used. Overall, all rules applied in MiPP
consistently revealed 4 genes that were highly robust classifiers,
and these genes were further confirmed using PCA. Interestingly,
two of the four "robust" predictive genes were not found to be
present when quantitative realtime RT-PCR was used to probe the RNA
sample. Finally a highly parsimonious biomarker was developed
herein, using MSI1 and LSI1 in combination with a subset of
biologically relevant genes present in the PAM-derived 62 gene TBB,
as well as from SAM analysis and evaluated this signature using
realtime RT-PCR; the 13 gene signature performed with perfect
accuracy to identify samples in the independent test set of this
case-control study. The observation that mean fold changes obtained
from realtime RT-PCR were not entirely identical with the results
from SAM analysis underlines the strength of molecular signature
analysis for the development of biomarkers, a classification
strategy that emphasizes differentially expressed gene expression
patterns rather than individual genes. Since the expression level
of an individual gene may vary across a population that shares the
same phenotype, the overexpression or downregulation of an entire
cluster of genes is more specific for a disease.
[0167] Based on these findings, it was concluded that both the
transcriptomic biomarker derived from PAM analysis, as well as the
parsimonious molecular signature that resulted from multiple
classification algorithms and testing for biological plausibility,
performed highly accurately and should be a clinically valuable
tool for the detection of myocarditis. While the more comprehensive
biomarker of 62 genes performed with slightly higher accuracy, the
13 genes molecular signature is more practical for clinical
application.
[0168] Since the original dataset was established by the inventors
in which the TBB was developed and was matched in a case-control
fashion, it was further evaluated if the molecular signature is
generalizable, or if it is possibly overfit to this particular
study design. It has been shown in the past that confounding
factors such as gender, age and therapy can affect gene expression.
When the TBB was applied in an additional validation set containing
samples from patients with an average EF that was twice as high as
the average EF of the original data set (65 vs 30%), the biomarker
performed with almost perfect accuracy.
[0169] Both molecular signatures will go into testing in a phase I
clinical trial, to further evaluate the diagnostic value of those
biomarkers in comparison to a combination of current diagnostic
tools, such as MRI, EKG, cardiac enzymes, viral screening and
auto-heart antibodies. Most likely, its addition to current
diagnostic standards will dramatically increase sensitivity for
myocarditis. The ability to detect inflammatory components, such as
involvement of the complement cascade or genes involved in cell
adhesion such as ITGB2 by microarray analysis may explain why this
technology is able to identify myocarditis with much greater
sensitivity at an earlier stage than standard histology, a method
that requires presence of inflammatory cells.
[0170] This study also addressed subtyping of inflammatory
cardiomyopathies. While the sample size of rare cardiomyopathies
was too small to finalize a minimal set of genes for clinical
application, it reveals highly robust molecular signatures that
distinguish patients with giant cell myocarditis, sarcoidosis, and
systemic lupus erythematosus noticeably from lymphocytic
myocarditis and IDCM. Interestingly, classification of peripartum
cardiomyopathy was less accurate, most likely because of multiple
factors interacting in this type of disease, ranging from
nonspecific changes such as replacement fibrosis to lymphocytic
infiltration.
[0171] The findings herein, that patients with giant cell
myocarditis share a gene expression profile that is highly distinct
from patients with cardiac sarcoidosis and that enables distinction
based on a single EMB, has important clinical implications. Due to
high histopathological similarity between giant cell myocarditis
and sarcoidosis, it may be that giant cell myocarditis may be a
subtype of the spectrum of cardiac sarcoidosis. Here it was shown
that these types of cardiomyopathy are clearly distinct from each
other on the molecular level. Importantly, one of the
differentially expressed genes in giant cell myocarditis vs
sarcoidosis was FCER1A, which has positive regulatory function in
type I hypersensitivity. While this finding may help in the future
to understand pathophysiology of these rare, hut clinically
important diseases, the ability to distinguish patients with giant
cell myocarditis from sarcoidosis has high clinical relevance for
risk assessment. Transplant-free survival is substantially greater
in cardiac sarcoidosis than in giant cell myocarditis, and giant
cell myocarditis may respond to treatment with monoclonal
antibodies against the CD3 receptor.
[0172] While the main goal of this study was to develop a highly
accurate biomarker to distinguish lymphocytic myocarditis from
IDCM, these results also provided insight into disease
pathophysiology on the molecular level. Among overexpressed genes
in myocarditis was CD8, involved in inflammation and binding and
reported to play a fundamental role in myocarditis. Surprisingly, a
pathway involving the TSH receptor was overexpressed in patients
with myocarditis, implicating potential pathophysiologic overlap
with inflammatory thyroid disease, a finding clinically established
for giant cell myocarditis (Graves'). There was overrepresentation
of patients, positive for the HLA-DQ1B locus in myocarditis vs
IDCM, suggesting possible susceptibility for lymphocytic
myocarditis in this group. Many transcripts, involving structural
proteins and muscle development (late cornified envelope 1 E,
collagen type I), were downregulated in myocarditis, possibly
explaining structural defects and consequent dilatation in patients
with this type of disease.
[0173] In short, a transcriptomic diagnostic biomarker was
discovered herein, derived from a single EMB, which identified
samples with lymphocytic myocarditis with very high accuracy. These
findings are highly relevant for a clinical application, since this
novel diagnostic tool exceeds sensitivity and specificity of any
technology that has been applied previously. The molecular
signature was highly robust and replicated multiple times by a
broad set of established classification algorithms. Validation in
two independent data sets revealed high diagnostic accuracy and
genes within the transcriptomic biomarker suggest biological
plausibility. Altogether, using this approach dramatically
increases the diagnostic accuracy of a single EMB, which may be of
critical importance to the development and allocation of emerging
specific therapies for inflammatory conditions of the heart.
TABLE-US-00001 TABLE 1 Overexpressed genes in patients with
myocarditis vs idiopathic dilated cardiomyopathy (q < 5%, FC
> 2) and their biological function Probe Set ID Gene Symbol Gene
Title GO biological process term 1552302_at FLJ77644, similar to
transmembrane protein 106A NA TMEM106A 1552553_a_at NLRC4 NLR
family, CARD domain containing 4 apoptosis, caspase activation,
defense response to bacterium, interleukin-1 beta secretion
1552584_at IL12RB1 interleukin 12 receptor, beta 1 cell surface
receptor linked signal transduction, positive regulation of cell
proliferation 1554899_s_at FCER1G Fc fragment of IgE, high affinity
I, positive regulation hypersensitivity, receptor for; gamma
polypeptide phagocytosis, engulfment, immunoglobulin mediated
immune response, positive regulation of interleukin-6 and 10 and
TNF production, positive regulation of mast cell cytokine
production 1555349_a_at ITGB2 integrin, beta 2 (complement
component 3 apoptosis, inflammatory response, cell adhesion,
receptor 3 and 4 subunit) leukocyte adhesion, integrin-mediated
signaling pathway 1559584_a_at C16orf54, chromosome 16 open reading
frame 54 NA hCG_1644884 1563245_at MGST1 microsomal glutathione
S-transferase 1 glutathione metabolic process 1565162_s_at ANXA2
annexin A2 skeletal development 1568126_at SPP1 Secreted
phosphoprotein 1 ossification, cell adhesion 1568574_x_at IFI30
interferon, gamma-inducible protein 30 oxidation reduction
201422_at CTSC cathepsin C proteolysis, immune response 201487_at
LAPTM5 lysosomal multispanning membrane protein 5 transport
201721_s_at CD14 CD14 molecule response to molecule of bacterial
origin, phagocytosis, apoptosis, inflammatory response 201743_at
CAPG capping protein (actin filament), protein complex assembly,
cell projection gelsolin-like biogenesis 201850_at PLTP
phospholipid transfer protein lipid metabolic process, transport
202075_s_at VAMP8 vesicle-associated membrane protein 8
vesicle-mediated transport (endobrevin) 202546_at LYN v-yes-1
Yamaguchi sarcoma viral related protein amino acid phosphorylation,
intracellular oncogene homolog signaling cascade, positive
regulation of cell proliferation, response to hormone stimulus,
erythrocyte differentiation, interspecies interaction between
organisms 202625_at ITGB2 integrin, beta 2 (complement component 3
apoptosis, inflammatory response, cell adhesion, receptor 3 and 4
subunit) leukocyte adhesion, integrin-mediated signaling pathway,
neutrophil chemotaxis 202803_s_at PCK2 phosphoenolpyruvate
carboxykinase 2 gluconeogenesis (mitochondrial) 202847_at CSF1R
colony stimulating factor 1 receptor protein amino acid
phosphorylation, signal transduction, transmembrane receptor
protein tyrosine kinase signaling pathway, multicellular organismal
development, cell proliferation 203104_at RASSF2 Ras association
(RalGDS/AF-6) domain family cell cycle, signal transduction,
negative member 2 regulation of cell cycle 203185_at RPS6KA1
ribosomal protein S6 kinase, 90 kDa, protein amino acid
phosphorylation, signal polypeptide 1 transduction, protein kinase
cascade 203379_at CD53 CD53 molecule signal transduction 203416_at
PLEK pleckstrin intracellular signaling cascade 203471_s_at SEMA4D
sema domain, immunoglobulin domain (Ig), NA transmembrane domain
(TM) and short cytoplasmic domain, (semaphorin) 4D 203528_at CD163
CD163 molecule acute-phase response, inflammatory response
203645_s_at PLA2G2A phospholipase A2, group IIA (platelets,
phospholipid metabolic process, lipid catabolic synovial fluid)
process 203649_s_at CXCL9 chemokine (C-X-C motif) ligand 9
chemotaxis, defense response, inflammatory response, cellular
defense response, G-protein coupled receptor protein signaling
pathway 203915_at CYBB cytochrome b-245, beta polypeptide
superoxide metabolic process, ion transport, inflammatory response,
superoxide release, innate immune response 203923_s_at IRF8
interferon regulatory factor 8 transcription, immune response,
myeloid cell differentiation 204057_at CD48 CD48 molecule defense
response 204118_at TYROBP TYRO protein tyrosine kinase binding
protein cellular defense response, intracellular signaling cascade
204122_at GLIPR1 GLI pathogenesis-related 1 NA 204222_s_at FCER1G
Fc fragment of IgE, high affinity I, receptor for; positive
regulation of hypersensitivity, positive gamma polypeptide
regulation of interleukin-10 and 6 and tumor necrosis factor
production, mast cell activation 204232_at PLEKHO2 pleckstrin
homology domain containing, family O NA member 2 204436_at CD44
CD44 molecule cell adhesion, cell-matrix adhesion 204490_s_at
SLC7A7 solute carrier family 7 (cationic amino acid amino acid
metabolic process, transport transporter, y+ system), member 7
204588_s_at STC1 stanniocalcin 1 cellular calcium ion homeostasis,
cell surface receptor linked signal transduction 204595_s_at CD52
CD52 molecule elevation of cytosolic calcium ion concentration,
respiratory burst 204661_at VSIG4 V-set and immunoglobulin domain
containing 4 negative regulation of interleukin-2 production,
negative regulation of T cell proliferation 204787_at IL10RA
interleukin 10 receptor, alpha NA 204912_at SASH3 SAM and SH3
domain containing 3 NA 204923_at TLR2 toll-like receptor 2 response
to molecule of fungal origin, MyD88- dependent toll-like receptor
signaling pathway, induction of apoptosis 204924_at CSTA cystatin A
(stefin A) peptide cross-linking 204971_at CCR1 chemokine (C-C
motif) receptor 1 chemotaxis, G-protein coupled receptor protein
signaling pathway, response to wounding 205098_at LCP2 lymphocyte
cytosolic protein 2 (SH2 domain immune response, transmembrane
receptor protein 205269_at containing leukocyte protein of 76 kDa)
tyrosine kinase signaling pathway, mast cell activation, cytokine
secretion 205270_s_at GZMA granzyme A (granzyme 1, cytotoxic T-
proteolysis, apoptosis, cleavage of lamin, lymphocyte-associated
serine esterase 3) immune response, cytolysis 205488_at CD86 CD86
molecule immune response, positive regulation of cell
proliferation, T cell activation 205685_at CD8A CD8a molecule
immune response, transmembrane receptor protein tyrosine kinase
signaling pathway, T cell activation 205758_at ITGAM integrin,
alpha M (complement component 3 cell adhesion, integrin-mediated
signaling receptor 3 subunit) pathway 205786_s_at LY86 lymphocyte
antigen 86 apoptosis, humoral immune response, cell proliferation
205859_at PTPN6 protein tyrosine phosphatase, non-receptor protein
amino acid dephosphorylation, apoptosis, type 6 response to
wounding 206687_s_at CCR2, FLJ78302 chemokine (C-C motif) receptor
2 chemotaxis, inflammatory response, cellular defense response,
JAK-STAT cascade, interspecies interaction between organisms
206978_at PTPRC protein tyrosine phosphatase, receptor negative
regulation of T cell mediated type, C cytotoxicity, positive
regulation of B cell proliferation, defense response to virus
207238_s_at SYK spleen tyrosine kinase serotonin secretion,
leukocyte adhesion neutrophil chemotaxis, interspecies interaction
between organisms, positive regulation of interleukin-3
biosynthetic process, positive regulation of B cell
differentiation, positive regulation of gamma-delta T cell
differentiation, positive regulation of alpha-beta T cell
differentiation 207540_s_at LILRB2 leukocyte immunoglobulin-like
receptor, immune response, cellular defense subfamily B (with TM
and ITIM domains), member 2 207697_x_at LCP1 lymphocyte cytosolic
protein 1 (L-plastin) actin filament bundle formation 208885_at
CORO1A coronin, actin binding protein, 1A phagocytosis 209083_at
HLA-DQB1 major histocompatibility complex, class II, antigen
processing and presentation of peptide or DQ beta 1 polysaccharide
antigen via MHC class II, immune response 209480_at DLK1 delta-like
1 homolog (Drosophila) multicellular organismal development
209560_s_at CD44 CD44 molecule (Indian blood group) cell adhesion,
cell-matrix adhesion 209835_x_at SPP1 secreted phosphoprotein 1
ossification, cell adhesion 209875_s_at AIF1 allograft inflammatory
factor 1 response to stress, inflammatory response, cell cycle
arrest, negative regulation of cell proliferation 209901_x_at C3AR1
complement component 3a receptor 1 chemotaxis, inflammatory
response, G-protein coupled receptor protein signaling pathway
209906_at CD300A CD300a molecule immune response, cell adhesion
209933_s_at NCF2 neutrophil cytosolic factor 2 cellular defense
response 209949_at LILRB2 leukocyte immunoglobulin-like receptor,
immune response, cellular defense response, cell subfamily B (with
TM and ITIM domains), surface receptor linked signal transduction
member 2 210146_x_at TLR1 toll-like receptor 1 inflammatory
response, macrophage activation, positive regulation of tumor
necrosis factor biosynthetic process, positive regulation of
interleukin-6 biosynthetic process 210176_at LAIR1
leukocyte-associated immunoglobulin-like immune response receptor 1
210644_s_at LILRB1 leukocyte immunoglobulin-like receptor, immune
response, response to virus subfamily B (with TM and ITIM domains),
member 1 211336_x_at TRBC1, TRBC2, T cell receptor beta constant 1,
T cell receptor immune response TRBV19 beta constant 2, T cell
receptor beta variable 19 211796_s_at CD44 CD44 molecule cell
adhesion, cell-matrix adhesion 212063_at PTPRC protein tyrosine
phosphatase, receptor type, C negative regulation of T cell
mediated cytotoxicity, cell surface receptor linked signal
transduction, T cell differentiation, positive regulation of B cell
proliferation, defense response to virus 212587_s_at 212588_at
HLA-DQA1, major histocompatibility complex, class II, DQ antigen
processing and presentation of peptide or HLA-DQA2 alpha 1, major
histocompatibility complex, class polysaccharide antigen via MHC
class II II, DQ alpha 2 212671_s_at hCG_1998957, major
histocompatibility complex, class II, DQ antigen processing and
presentation of peptide or HLA-DQB1/B2, beta 1 and 2; DR beta1, 2,
3, 4 and 5 polysaccharide antigen via MHC class II HLA-DRB1/2/3/4/5
212999_x_at AIF1 allograft inflammatory factor 1 response to
stress, inflammatory response, cell cycle arrest 213095_x_at DOCK2
dedicator of cytokinesis 2 actin cytoskeleton organization and
biogenesis, lymphocyte chemotaxis 213160_at HSPA6 heat shock 70 kDa
protein 6 (HSP70B') response to stress 213418_at RNASE6
ribonuclease, RNase A family, k6 RNA catabolic process, defense
response 213566_at RAC2 ras-related C3 botulinum toxin substrate 2
(rho chemotaxis, positive regulation of cell family, small GTP
binding protein Rac2) roliferation, regulation of respiratory burst
213603_s_at MYO1F myosin IF NA 213733_at HLA-DQA1 major
histocompatibility complex, class II, DQ antigen processing and
presentation of peptide alpha 1 or polysaccharide antigen via MHC
class II 213831_at LYZ lysozyme (renal amyloidosis) tRNA
aminoacylation for protein translation, inflammatory response,
defense response to bacterium 213975_s_at LOC648998 similar to
Neutrophil cytosol factor 1 (NCF-1) NA (Neutrophil NADPH oxidase
factor 1) (47 kDa
neutrophil oxidase factor) (p47-phox) (NCF-47K) (47 kDa autosomal
chronic granulomatous disease protein) (NOXO2) 214084_x_at CD163
CD163 molecule acute-phase response, inflammatory response
215049_x_at AIF1 allograft inflammatory factor 1 response to
stress, inflammatory response, cell cycle arrest, negative
regulation of cell proliferation 215051_x_at ADA adenosine
deaminase response to hypoxia, adenosine catabolic process, T cell
activation 216705_s_at FCGR1A, FCGR1C Fc fragment of IgG, high
affinity Ia, Ic, phagocytosis, engulfment receptor (CD64)
216950_s_at GLUL glutamate-ammonia ligase (glutamine glutamine
biosynthetic process, nitrogen synthetase) compound metabolic
process 217202_s_at SNX10 sorting nexin 10 transport, cell
communication 218404_at MAFB v-maf musculoaponeurotic fibrosarcoma
transcription oncogene homolog B (avian) 218559_s_at CCDC109B
coiled-coil domain containing 109B NA 218802_at BIN2 bridging
integrator 2 NA 219191_s_at DOCK10 dedicator of cytokinesis 10 NA
219279_at SLAMF8 SLAM family member 8 NA 219386_s_at SIGLEC1 sialic
acid binding Ig-like lectin 1, inflammatory response, cell
adhesion, cell- sialoadhesin matrix adhesion, cell-cell adhesion
219519_s_at 1-Mar membrane-associated ring finger (C3HC4) 1 NA
219574_at MS4A4A membrane-spanning 4-domains, subfamily A, signal
transduction member 4 219607_s_at MS4A6A 219666_at GAL3ST4
galactose-3-O-sulfotransferase 4 sulfur metabolic process,
cell-cell signaling, biosynthetic process 219815_at PSTPIP2
proline-serine-threonine phosphatase NA interacting protein 2
219938_s_at TLR7 toll-like receptor 7 inflammatory response,
positive regulation of interferon-gamma biosynthetic process,
positive regulation of interleukin-8 biosynthetic process, defense
response to virus 220146_at COTL1 coactosin-like 1 (Dictyostelium)
carbohydrate metabolic process 221059_s_at NPL N-acetylneuraminate
pyruvate lyase carbohydrate metabolic process (dihydrodipicolinate
synthase) 221210_s_at SH3BGRL3 SH3 domain binding glutamic
acid-rich protein NA like 3 221269_s_at PYCARD PYD and CARD domain
containing proteolysis, apoptosis, tumor necrosis factor- mediated
signaling pathway, positive regulation of interleukin-1 beta
secretion 221666_s_at CLEC7A C-type lectin domain family 7, member
A phagocytosis, recognition, inflammatory response, T cell
activation, defense response to protozoan 221698_s_at OBFC2A
oligonucleotide/oligosaccharide-binding fold NA containing 2A
222872_x_at CENTA2 centaurin, alpha 2 heart development 222876_s_at
MS4A7 membrane-spanning 4-domains, subfamily A, signal transduction
member 7 223343_at 223344_s_at MS4A6A membrane-spanning 4-domains,
subfamily A, signal transduction member 6A 223922_x_at 224356_x_at
MS4A4A membrane-spanning 4-domains, subfamily A, signal
transduction member 4 224357_s_at COTL1 coactosin-like 1
(Dictyostelium) NA 224583_at BCAT1 branched chain aminotransferase
1, cytosolic G1/S transition of mitotic cell cycle, metabolic
process, cell proliferation, amino acid biosynthetic process
225285_at C1QC complement component 1, q subcomponent, phosphate
transport, complement activation, C chain classical pathway
225353_s_at CTSC cathepsin C proteolysis, immune response 225646_at
CTSC 225647_s_at BCAT1 branched chain aminotransferase 1, cytosolic
G1/S transition of mitotic cell cycle, metabolic process, cell
proliferation, amino acid biosynthetic process 226517_at MPEG1
macrophage expressed gene 1 NA 226818_at 226841_at FYB FYN binding
protein (FYB-120/130) carbohydrate metabolic process, protein amino
acid phosphorylation, immune response, signal transduction
227266_s_at RILPL2 Rab interacting lysosomal protein-like 2 NA
227983_at OSR1 odd-skipped related 1 (Drosophila) heart development
228399_at C1orf162 chromosome 1 open reading frame 162 NA 228532_at
LILRB1 Leukocyte immunoglobulin-like receptor, immune response,
response to virus subfamily B (with TM and ITIM domains), member 1
230741_at MRO maestro NA 231358_at CTSS cathepsin S proteolysis,
immune response 232617_at DOCK8 dedicator of cytokinesis 8 NA
232843_s_at OBFC2A oligonucleotide/oligosaccharide-binding fold NA
containing 2A 233085_s_at PARVG parvin, gamma cell adhesion,
cell-matrix adhesion 234987_at CPM carboxypeptidase M proteolysis,
anatomical structure morphogenesis 235019_at HAVCR2 hepatitis A
virus cellular receptor 2 NA 235458_at CCL18 chemokine (C-C motif)
ligand 18 (pulmonary chemotaxis, inflammatory response and
activation-regulated) 32128_at CD52 CD52 molecule elevation of
cytosolic calcium ion concentration, respiratory burst 34210_at
MAFF v-maf musculoaponeurotic fibrosarcoma response to stress,
regulation of transcription oncogene homolog F (avian) 36711_at
SIGLEC1 sialic acid binding Ig-like lectin 1, inflammatory
response, cell adhesion, cell- sialoadhesin matrix adhesion
TABLE-US-00002 TABLE 2 Downregulated genes in patients with
myocarditis vs idiopathic dilated cardiomyopathy (q < 5%, FC
> 2) and their biological function Probe Set ID Gene Symbol Gene
Title GO biological process term 1552411_at DEFB106A/B defensin,
beta 106A defence response, defense response to bacterium
1556721_at FLJ33706 hypothetical protein FLJ33706 NA 1559224_at
LCE1E late cornified envelope 1E keratisization 1562256_at NLRP1
NLR, family pynn domain containing 1 induction of apoptosic,
caspase activation, defense response 1562257_x_at 1562785_at HERC6
Hest domain and RLD 6 protein modification process 1564281_at
LOC285708 hypothetical protein LOC285708 nucleotide and nucleic
acid metabolic process, nervous system development 1564362_x_at
ZNF843 zinc finger protein 243 NA 1569568_at NA NA NA 1569569_x_at
NA NA NA 213609_s_at SEZ6L seizure related 6 homolog (mouse)-like
NA 213791_at PENK proenkephalin behavioral fear response, signal
transduction, neuropeptide signaling pathway, sensory perception of
pain 224269_s_at GDA guanine deaussinase nucleotide and nucleic
acid membolic process, nervous system development 231623_s_at NA NA
NA 243909_x_at GUSBL2 glucuronidase, beta-like 2 NA 244891_x_at NA
NA NA
TABLE-US-00003 TABLE 3 Overexpressed pathways in patients with
myocarditis vs idiopathic dilated cardiomyopathy Total Root Network
GO Processes nodes nodes p-Value zScore MafB, MafF, MHC system
development (66.0% 2.241e-13); response to stimulus (74.5%; 50 10
2.43E-17 29.34 class II, CD44, BCAT1 1.751e-12), multicellular
organismal development (68.1%; 7.914e-12), (Homo sapiens) organ
development (55.3%; 2.289e-11), positive regulation of cellular
process (51.1%, 9.353e-11) CCR1, BCAT1, ADA, response to external
stimulus (53.8%; 2.384e-09), intracellular signaling 50 7 1.87E-12
24.29 Annexin II, Pleckstrin cascade (57.7%; 1.087e-08), behavior
(38.5%; 4.275e-08), response to (Homo sapiens) chemical stimulus
(53.8%; 8.258e-08), MAPKKK cascade (26.9%; 1.123e-07) p47-phox,
CCR2, p67- protein kinase cascade (48.8%; 2.208e-20), intracellular
signaling 50 7 1.53E-11 21.11 phox, Pleckstrin, IL-12 cascade
(68.3%; 6.669e-18); response to chemical stimulus (61.6%; receptor
(Homo 1.232e-14), regulation or cell migration (29.3%; 3.332e-14),
MAPKEK sapiens) cascade (31.7%; 3.194e-14) C1q, CD44, CD14,
cell-matrix adhesion (30.4%; 2.499e-10) cell-substrate adhesion 24
4 2.85E-07 16.72 SLAP-130(ADAP), (30.4%; 4.574e-10), positive
regulation of biological process (69.6%; alpha-4/beta-1 integrin
1.047e-09), cell adhesion (47.8%; 2.037e-03), biological adhesion
(Homo sapiens) (47.8%; 3.037e-08) Plastin, IRT-1 (Homo actin
filament bundle formation (100.0%; 1.902e-05), actin filament 2 2
5.36E-06 29.3 sapiens) organization (100.0%; 5.224e-05), actin
cytoskeleton organization (100.0%; 4.702e-04), achu filament-based
process (100.0%; 5.330e-04), macrophage activation (50.0%,
2.438e-03) CD163, HP/HB acute inflammatory response (100.0%;
1.664e-04), response to L- 2 1 4.64E-03 14.62 complex (Homo
ascorbic acid (50.0%; 4.879e-04), nitric oxide transport (50.0%;
4.879e-04), sapiens) inflammatory response (100.0%; 1.161e-03),
response to magnesium ion (50.0% 1.341e-03) Complement complement
activation, classical pathway (100.0%; 3.660e-03), ghal 8 1
1.16E-02 9.18 component C1, cell differentiation (100.0%;
3.904e-03), humoral immune response Complement C4 = mediated by
circulating immunoglobulin (100.0%; 4.026e-03); Complement
activation of plasma proteins during acute inflammatory response
component C4a + (100.0%; 4.819e-03), complement activation (100.0%
4.819e-03) Complement component C4b, Complement C2 = Complement
component C2a + Complement component C2b, Complement component C4a,
C4a (Homo sapiens) PLTP, ABCA1, response to drug (60.0% 7.494e-05),
platelet dense granule 19 1 3.88E-02 4.84 CREB1, Cholesterol
organization and biogenesis (20.0%; 3.050e-04), response to vitamin
K extracellular region, (20.0%; 3.050e-04), response to menaquinane
(20.0%; 3.050e-04), Cholesterol + ATP + positive regulation of
growth (40.0%, 3.354e-04) H(.2)O = Cholesterol + ADP + PO(.4)(3-)
(Homo sapiens)
TABLE-US-00004 TABLE 4 Molecular signature that discriminates giant
cell myocarditis from idiopathic dilated cardiomyopathy Gene Probe
Set ID Symbol Gene Title Go Biological Process Term 210067_at AQP4
aquaporin 4 Transport, water transport, nervous system development,
excretion 221212_x_at PBRM1 polybromo 1 chromatin remodeling,
regulation of transcription, mitosis, chromosin modification
227145_at LOXL4 lysyl oxidase-like-4 oxidation reduction 228329_at
DAB1 disabled homolog 1 (Drosophila) multicellular organismal
development, nervous system development, cell differentiation
231577_s_at GBP1 guanylate binding protein 1, immune response
interferon-inducible, 67 kDa 231906_at HOXD8 Homeobox D8 regulation
of transcription, multicellular organsimal development,
determination of anteriorposterior axis, embryo, regulation of
transcription 235334_at ST6GALNAC3 ST6 (alpha-N-acetyl-neurammyl-
protein amino acid glycosylation 2,3-beta-galactosyl-1,3)-N-
acetylgalactosaminide alpha-2,6- sialyluansferase 3 237783_at
PLAC8L1 PLAC8-like-1 NA
TABLE-US-00005 TABLE 5 Molecular signature that discriminates
sarcoidosis from idiopathic dilated cardiomyopathy Gene Probe Set
ID Symbol Gene Title Go Biological Process Term 1552974_at NA NA NA
1553781_at ZC3HAV1L zinc finger CCCH-type, antiviral 1-like NA
1554478_a_at HEATR3 HEAT repeat containing 3 NA 1556760_a_at NA NA
NA 1556883_a_at LOC440896 hypothetical gene LOC440896 NA 1557717_at
LOC338862 hypothetical protein LOC338862 NA 1560144_at NA NA NA
1560683_at BCL8 B-cell CLL/lymphoma 8 NA 1560624_x_at BCL8 B-cell
CLL/lymphoma 8 NA 1561543_at NA NA NA 1562035_at NA NA NA
1563054_at NA NA NA 1563452_at KIAA0241 KIAA0241 NA 1564107_at NA
NA NA 1564733_at NA NA NA 1565728_at NA NA NA 1566550_at NA NA NA
1568589_at NA NA NA 201291_s_at TOP2A topoisomerase (DNA) II alpha
170 kDa DNA metabolic process, DNA replication, response to DNA
damage stimulus, apoptotic chromosome condensations, positive
regulation of viral genome replication, positive regulation of
retroviral genome replication 204666_s_at RP5-1000E10.4 exppressor
of IKK epsilon NA 208536_s_at BCL2L11 BCL2-like 11 (apoptosis
facilirator) Induction of apoptosis, activation of pro-apoptotic
gene products 209371_s_at SH3BP2 SH3-domain binding protein 2
signal transduction 215512_at 6-Mar membrane-associated ring finger
(C3HC4) 6 NA 216947_at DES desmin muscle contraction, cytoskeleton
organization and biogenesis, regulation of heat contraction
217292_at MTMR7 myotubularin related protein 7 protein amine acid
dephosphorylation, phospholipid dephosphorylation 218554_a_at ASH1L
ash1 (absent, small, or homeotic)-like DNA packaging, regulation of
transcription, (Drosophila) transcription from RNA polymerase II
promoter, cell- cell signalling, chromatin modification 218585_s_at
DTL denticleless homolog (Drosophila) DNA replication, response to
DNA damage stimulus 219258_at TIPIN TIMELESS interacting protein
DNA replication checkpoint, response to DNA damage stimulus, cell
cycle, mitosis, positive regulation of cell proliferation, intra-S
DNA damage checkpoint, replication fork protection, cell division
219735_s_at TFCP2L1 transcription factor CP2-like 1 Negative
regulation of transcription from RNA polymerase II promoter, cell
morphogenesis, epithelial cell maturation, reguistion of
transcription, steroid biosynthetic process, determination of adult
life span 219918_s_at ASPM sup (abnormal spindle) homolog, cell
cycle, mitosis, cell division microcephaly associated (Drosophila)
220085_at HELLS helicase, lymphoid-specific methylation-dependent
chromatin silencing, regulation of transcription, cell cycle,
mitosis, multicellular organismal development, centromeric
heterochromatin formation, lymphocyte proliferation 220735_s_at
SENP7 SUMO1/rentrin specific peptidase 7 Proteolysin, protein
sumoylation 220930_s_at MGC5590 hypothetical protein MGC5590 NA
221212_x_at PERM1 polybroma 1 chromatin remodeling, regulation of
transcription, DNA- dependent, mitosis, chromatin modification
221268_s_at SGPP1 splingonine-1-phosphate phosphatase 1
splingolipid metabolic process, splinganine-1- phosphate metabolic
process, apoptosis 221969_at NA NA NA 223700_at MND1 meiotic
nuclear divisions 1 homolog (S. cerevisiae) DNA recombination,
meiosis 223865_at SOX6 SRY (sex determining region Y)-box 6
establishment or maintenance of chromain architecture, regulation
of transcription, multicellular organismal development, muscle
development 224424_x_at LOC440888 ARP3 actin-related protein 3
homolog E regulation of actin filament polymerization psendogene
224426_s_at LOC440888 ARP3 actin-related protein 3 homolog B
regulation of actin filament polymerization psemdogene 232453_at NA
NA NA 233785_at NA NA NA 235588_at ESCO2 establishment of cohesion
1 homolog 2 (S. cerevisiae) DNA repair, cell cycle 235661_at NA NA
NA 233899_at CA13 carbonic anhydrase XIII one-carbon compound
metabolic process 236628_at NA NA NA 236740_at NA NA NA 237289_at
CREB1 cAMP responsive element binding protein 1 regulation of
transcription, protein amino acid phosphorylation, signal
transduction, interspecies interaction between organisms
238370_x_at RPL22 Ribosomal protein L22 Translation, translational
elongation 233375_at 239486_at NA NA NA 239899_at RNF145 Ring
finger protein 145 NA 241922_at NA NA NA 242784_at NA NA NA
242939_at TFDP1 transcription factor Dp-1 S phase of mitotic cell
cycle, regulation of transcription, apoptosis, cell proliferation,
epidermis development 244356_at NA NA NA 244605_at NA NA NA
37892_at COL11A1 collagen type XI, alpha 1 carriage condensation,
phosphate transport, cell adhesion, extracellular matrix
organisation and biogenesis
TABLE-US-00006 TABLE 6 Molecular signature that discriminates
peripartum cardiomyopathy from idiopathic dilated cardiomyopathy
Gene Probe Set ID Symbol Gene Tide Go Biological Process Term
1553972_a_at CBS cystathionine-beta synthase cysteine metabolic
process 1557833_at NA NA NA 1550395_at NA NA NA 201909_at
LOC190133662, hypothetical protein translational elongation RPS4Y1
LOC109133662, nbosamai protein S4, Y-linked 1 204409_s_at EIF1AY
eukaryotic translation mitiation translational mitiation 204410_at
factor 1A, Y-linked 205000_at DDX3Y DEAD (Asp-Gln-Als-Asp) box NA
205001_s_at polypeptide 3, Y-linked 205033_s_at DEFA1, DEFA3,
defeasin, alpha 1, defeasin, xenobiotic metabolic process,
chemotaxis, defense response, LOC728358 alpha 3,
neutrophil-specific immunse response, response to virus, defense
response to defensin, alpha 1 bacterium, defense response to fungus
205048_s_at PSPH phosphoserine phosphatese L-serine metabolic
process 205609_at ANGPT1 ongiopeietin 1 Angiogenesit, signal
transduction, multicellular organismal development, cell
differentiation 206624_at LOC100130216, hypothetical protein
ubiquitin-dependent protein carbolic process USP9Y LOC100130215,
ubiquitin specific peptidase 9. Y-linked (fat facets-like,
Drosophsla) 206700_s_at JARID1D jumonji, AT rich interactive
chromatin modification, oxidation reduction domain 1D 207063_at
CYorf14 chromosome Y open reading NA frame 14 203067_x_at
LOC100130224, hypothetical protein chromatin modification,
oxidation reduction UTY LOC100130224, ubiquitously transcribed
tetrstricepeptide repeat gene, Y-linked 209771_x_at CD24 CD24
molecule response to hypoxis, cell activation, regulation of
cytokine and chemokine mediated signaling pathway, response to
molecule of bacterial origin, immune response-regulating cell
surface receptor signaling pathway, elevation of cytosolic calcium
ion concentration, neuromuscular synaptic transmission, induction
of apoptosis by intracellular signals. Wnt receptor signaling
pathway, cell-cell adhesion, positive regulation of activated T
cell proliferation 211018_at LSS lnosterol synthase (2,3- steroid
biosynthetic process, metabolic process, steroid
oxidosqualene-lanotherol metabolic process, lipid biosynthetic
process cyclase) 211149_at LOC100130224, hypothetical protein
chromatin modification, oxidation reduction UTY LOC100130224,
ubiquitously transcribed tetratricopeptide repeat gene, Y-linked
213768_s_at OLFM4 olfactomedin 4 cell adhesion 212816_s_at CBS
cystathionine-beta-synthase cysteine metabolic process 212906_at
GRAMD1B GRAM domain containing 1B NA 214131_at CYorf15B chromosome
Y open reading NA frame 15B 214218_s_at XIST X (inactive)-specific
transcript NA (non-protein coding) 214983_at TTTY15 testis-specific
transcript, Y- NA linked 15 216758_at NA NA NA 219938_s_at PSTPIP2
proline-serine-threonine NA phosphatese interacting protein 2
221722_x_at XIST X (inactive)-specific transcript NA (non-protein
coding) 223645_s_at CYorf15B chromosome Y open reading NA
223646_s_at frame 15B 224293_at TTTY10 testis-specific transcript,
Y- NA linked 10 224588_at XIST X (inactive)-specific transcript NA
224589_at (non-protein coding) 224590_at 22761_at 227742_at CLIC6
chloride intracellular channel 6 Transport, ion transport, chloride
transport 228194_s_at SORCS1 sortilin-related VPS10 domain
neuropeptide signaling pathway cintaining recepter 1 228492_at
LOC100130216, hypothetical protein ubiquitin-dependent protein
carabolic process USP9Y LOC100130216, ubiqutin specific peptidase
9, Y-linked (fat facets-like, Drosophila) 229160_at MUM1L1 melanoma
associated antigen NA (mutated) 1-like 1 229534_at ACOT4 acyl-CoA
thioesterase 4 very-long-chain fasty acid metabolic process,
long-chain fatty acid metabolic process, acyl-CoA metabolic
process, lipid metabolic process, acyl-CoA metabolic process,
unsaturated monocarboxylic acid metabolic process, unsaturated
monocarboxylic acid metabolic process, dicarboxylic acid metabolic
process, short-chain fatty acid metabolic process 239104_s_at TPPP
tubulin polymerization microtubule bundle formation, positive
regulation of protein promossing protein complex assembly,
microtubule polymerization 230760_at LOC100130829, hypothetical
protein regulation of transcription ZFY LOC100130829, zinc finger
protein, Y-linked 231592_at TSIK X (inactive)-specific transcript,
NA antisense (non-protein coding) 232365_at SIAH1 seven in absentin
homolog 1 Proteolysis, ubiquitin-dependent protein carabolic
process, (Drosophila) apoptosis, cell cycle, multicellular
organismal development, nervous system development, axon guidance,
cell differentiation 232618_at CYorf15A chromosome Y open reading
NA frame 15A 233176_at NA NA NA 235334_at ST6GALNAC3 ST6
(alpha-N-acetyl- protein amino acid glycosylation
neuraminyl-2,3-beta- galactotyl-1,3)-N- acetylgalactosaminide
alpha- 2,5-sialyltransferase 3 235446_at NA NA NA 235942_at
LOC401629, LOC401629, LOC401630 LOC401630 236694_at CYorf15A
chromosome Y open reading NA frame 15A 239568_at PLEKHH2 plecketrin
homology domain NA containing, family H (with MyTH4 domain) member
2 239584_at NA NA NA 239677_at NA NA NA 242316_at NA NA NA
243610_at C9orf135 chromosome 9 open reading NA frame 135 244482_at
NA NA NA 266_s_at CD24 CD24 molecule response to hypoxis, cell
activation, regaistion of cytokine and chemokine mediated signaling
pathway, response to molecule of bacterial orgin, immune
response-regulating cell surface receptor signaling pathway,
elevation of cytosolic calcium ion concentration, neuromuscular
synaptic transmission, induction of apoptosis by intracellular
signals, Wnt receptor signaling pathway, cell-cell adhesion,
positive regulation of activated T cell proliferation
TABLE-US-00007 TABLE 7 Molecular signature that discriminates
systemic lupus erythematosus from idiopathic dilated cardiomyopathy
Probe Set ID Gene Symbol Gene Title Go Biological Process Term
1552946_at ZNF114 zinc finger protein 114 Regulation of
transcription 1553607_at C21orf109 chromosome 21 open reading frame
NA 109 1555485_s_at FAM153B family with sequence similarity 153, NA
member B 1558882_at LOC401233 cofactor required for Tat activation
NA of HIV-1 transcription 1561012_at NA NA NA 1566518_st NA NA NA
1569539_at NA NA NA 1569794_st NA NA NA 207781_s_at ZNF711 zinc
finger protein 711 Regulation of transcription 222375_at NA NA NA
229288_at NA NA NA 229523_at TTMA Two transmembrane domain family
NA member A 235803_at NA NA NA 238533_at EPHA7 EPH receptor A7
protein amino acid phosphorylation, transmembrane receptor protein
tyrosine kinase signaling pathway 238755_at NA NA NA 240783_at NA
NA NA 240903_at NA NA NA 242641_at NA NA NA 243012_at NA NA NA
244626_at NA NA NA 244636_at NA NA NA
TABLE-US-00008 TABLE 8 Molecular signature that differentiates
giant cell myocarditis from lymphocytic myocarditis Gene Go
Biological Probe Set ID Symbol Gene Title Process Term 1563283_at
NA NA NA 204477_at RABIF RAB Transpost, membrane interacting
fusion, small GTPase factor mediated signal transduction, protein
transport 205275_at GTPBP1 GTP binding immune response, protein 1
signal transduction, cell redox homeostasis 214313_s_at EIF5B
Eukaryotic regulation of translation translational initiation
initiation factor 5B
TABLE-US-00009 TABLE 9 Molecular signature that differentiates
sarcoidosis from lymphocytic myocarditis Gene Probe Set ID Symbol
Gene Title go biological process term 204477_at RABIF RAB
interacting factor Transport, membrane fusion, small GTPase
mediated signal transduction, protein transport 205275_at GTPBP1
GTP binding protein 1 immune response, signal transduction, cell
redox homeostasis 214313_s_at EIF5B Eukaryotic translation
Translation, regulation of translational initiation initiation
factor 5B 224500_s_at MON1A MON1 homolog A (yeast) NA 236093_at NA
NA NA 243564_at PDE1C phosphodiesterase 1C, signal transduction
calmodulin-dependent 70 kDa
TABLE-US-00010 TABLE 10 Molecular signature that differentiates
peripartum cardiomyopathy from lymphocytic myocarditis Probe Set ID
Gene Symbol Gene Title Go Biological Process Term 1563283_at NA NA
NA 205275_at GTPBP1 GTP binding protein 1 immune response, signal
transduction, cell redox homeostasis 207300_s_at F7 coagulation
factor VII (serum Proteolysis, blood coagulation, extrimic pathway
profrombin converson accelerator) 214313_s_at EIF5B Eukaryotic
translation initiation factor 5B regulation of translational
initiation 214473_x_at PMS2L3 postmeiotic segregation increased
2-like 3 mismatch repair, regulation of transcription 227509_x_at
NA NA 228232_s_at VSIG2 V-set immunoglobulin domain NA containing 2
230731_x_at ZDHHC8 zinc finger, DHHC-type containing 8 NA
232586_x_at LOC100133315 Similar to hCG1640299 single strand break
repair 236093_at NA NA NA 237867_s_at PID1 phosphotyrosine
interaction domain NA containing 1 243564_at PDE1C
phosphodiesterase 1C, calmodulin. signal transduction dependent 70
kDa
TABLE-US-00011 TABLE 11 Molecular signature that differentiates
systemic lupus erythematosus from lymphocytic myocarditis Gene
Probe Set ID Symbol Gene Title Go Biological Process Term
1556205_at NA NA NA 202179_at BLMH blecomycin hydrolase
Proteolysis, response to toxin, response to drug 203134_at PICALM
phosphatidylinositol protein complex assembly, endocytosis,
receptor-mediated endocytosis, receptor- binding clathrin mediated
endocytosis, vesicle-mediated transport, clathrin coat assembly
assembly protein 203540_at GFAP glial fibrillary acidic NA protein
203554_s_at DNASEIL3 deoxyribonuclease I- DNA metabolic process.
DNA catabolic process, DNA fragmentation during like 3 apoptosis
205673_s_at ASB9 ankyrin repeat and intracellular signaling escade
SOCS box-containing 9 205794_s_at NOVA1 neuro-encological RNA
processing, synaptic transmission, locomotory behavior, RNA
splicing ventral antigen 1 209220_at GPC3 glypican 3 anatomical
structure morphogenesis 209304_s_at GADD45B growth arrest and DNA-
activation of MAPKKK activity, negative regulation of protein
kinase activity, damage-inducible, beta apoptosis, response to
stress, multicellular organismal development cell differentiation
209540_at IGF1 insulin-like growth skeletal development, DNA
replication, anti-apoptosis, muscle development, factor 1
(somatomedin positive regulation of cell proliferation, satellite
cell maintenance involved in C) skeletal muscle regeneration,
muscle hypertrophy, myotube cell development positive regulation of
tyrosine phosphorylation of Stat5 protein, myoblast
differentiation, positive regulation of fibroblast proliferation
209923_s_at BRAP BRCA1 associated negative regulation of signal
transduction protein 212173_at AK2 adenyiate kinase 2 nucleobase,
nucleoside, nucleotide and nucleic acid metabolic process 213496_at
LPPR4 plasticity related gene 1 NA 214358_at DNAJB12 DnaJ (Hep40)
homolog, protein folding subfamily B, member 12 216269_s_at ELN
elastin DNA repair, respiratory geneous exchange, blood
circulation, cell proliferation organ morphogenesis 217950_at NOSIP
nitric oxide synthase protein ubiquitinarion, negative regulation
of catalytic activity, negative regulation interacting protein of
nitric-oxide synthase activity 218180_s_at EPS8L2 EPS8-like 2 NA
220117_at ZNF385D zinc finger protein 385D NA 220941_s_at C21orf91
chromosome 21 open NA reading frame 91 222002_at C7orf26 Chromosome
7 open NA reading frame 26 222879_s_at POLH polymerase (DNA DNA
synthesis during DNA repair directed), etc 223574_x_at PPP2R2C
protein phosphatase 2 signal transduction (formerly 2A), regulatory
subunit B, gamma isoform 223586_at ARNTL2 aryl hydrocarbon
regulation of transcription, signal transduction, entrainment of
circadian clock receptor nuclear translocator-like 2 230974_at
DDX19B DEAD (Asp-Glu-Ala- mRNA export from, intracellular protein
transport across a membrane As) box polypeptide 19B 233298_at
C13orf38 chromosome 13 open regulation of transcription,
multicellular organismal development, cell SOHLH2 reading frame 38,
differentiation spermatogenesis and oogenesis specific basic
helix-loop-helix 2 238151_at NA NA NA 243076_x_at GLI4 GLI-Kruppel
family NA member GLI4
TABLE-US-00012 TABLE 12 Molecular signature to distinguish giant
cell myocarditis from sarcoidosis Probe Set ID Gene Symbol Gene
Title Go Biological Process Term 1553894_at CCDC122 coiled-coil
domain containing 122 NA 1557311_at LOC100131354 Hypothetical
protein LOC100131354 NA 1557996_at POLR2J4 polymerase (RNA) II (DNA
directed) transcription polypeptide J4, pseadogene 1558450_at NA NA
NA 1559227_s_at VHL von Hippel-Lindau tumor suppressor negative
regulation of transcription from RNA polymerase II promoter, cell
morphogenesis, proteolysis, anti-apoptosis, response to stress,
negative regulation of cell proliferation, regulation of cell
differentiation. negative regulation of cell cycle 1561789_at NA NA
NA 1569312_at NA NA NA 205238_at CXorf34 chromosome X open reading
frame 34 NA 211734_s_at FCER1A Fc fragment of IgE, high affinity I,
positive regulation of type I hypersensitivity, serotonin receptor
for, alpha polypeptide secretion, cell surface receptor linked
signal transduction, leukotriene biosynthetic process, positive
regulation of mast cell degranulation, positive regulation of
interleukin-3 biosynthetic process, positive regulation of
granulocyte macrophage colony-stimulating factor biosynthetic
process 218669_at RAP2C RAP2C, member of RAS oncogene small GTPase
mediated signal transduction family 225207_at PDK4 pyruvate
dehydrogenase kinase, isozyme 4 carbohydrate metabolic process,
glucose metabolic process, signal transduction, phosphorylation
231114_at SPATA22 spermatogenesis associated 22 NA 231418_at NA NA
NA 231819_at NA NA NA 231956_at KIAA1618 KIAA1618 NA 233927_at NA
NA NA 239151_at CTGLF6 centaurin, gamma-like family, member 6
regulation of ARF GTPase activity 241788_x_at NA NA NA 242691_at NA
NA NA
TABLE-US-00013 TABLE 13 Baseline conditions of patients with
idiopathic dilated cardiomyopathy and lymphocytic myocarditis
Idiopathic dilated cardiomyopathy Myocarditis (n = 32) (n = 16) Age
48 (.+-.3) 45 (.+-.6) Male, n (%) 11 (38) 11 (69) NYHA, n (%) I 9
(28) 4 (25) II 10 (31) 3 (19) III 13 (59) 8 (50) IV 3 (9) 1 (6) LV
EF, % 26 .+-. 2 33 .+-. 4 LVIDD, cm 5 .+-. 0.3 5 .+-. 0.2 PAP, mmHg
Systolic 38 .+-. 3 37 .+-. 3 Diastolic 18 .+-. 2 15 .+-. 2 PCWP,
mmHg 15 .+-. 2 12 .+-. 2 Systolic BP, mmHg 128 .+-. 5 119 .+-. 5
Diastolic BP, mmHg 76 .+-. 2 70 .+-. 4 Medications, n (%)
B-Antagonist 20 (62) 9 (56) ACE inhibitor 20 (62) 14 (88)
Aldosterone antagonist 4 (13) 1 (6) Diuretic 14 (64) 13 (81)
Intravenous inotropic therapy NA NA Statistics: Student t-test,
Fisher Exact test; .+-. refers to standard error of the mean
TABLE-US-00014 TABLE 14 Transcriptomic diagnostic biomarker for
detection of patients with myocarditis: 62 genes Probe Set ID Gene
Symbol Gene Title GO biological process term 1552302_at FLJ77644,
TMEM106A similar to transmembrane protein 106A, NA transmembrane
protein 106A 1552310_at C15orf40 chromosome 15 open reading frame
40 NA 1553212_at KRT78 keratin 78 NA 1555349_s_at ITGB2 integrin,
beta 2 (complement component 3 apoptosis, inflammatory response,
receptor 3 and 4 subunit) leukocyte adhesion 1555878_at RPS24
Ribosomal protein S24 translation 1556033_at NA NA NA 1556507_at NA
NA NA 1558605_at NA NA NA 1559224_at LCE1E late cornified envelope
1E keratinization 1562785_at HERC6 Hect domain and RLD 6 protein
modification process 1565662_at NA NA maintenance of
gastrointestinal epithelium 1565830_at NA NA NA 202375_at SEC24D
SEC24 related gene family, member D (S. cerevisiae) transport,
intracellular protein transport 202445_s_at NOTCH2 Notch homolog 2
(Drosophila) cell fate determination 203741_s_at ADCY7 adenylate
cyclase 7 cAMP biosynthetic process, signal transduction
204222_s_at GLIPR1 GLI pathogenesis-related 1 NA 206052_s_at SLBP
stem-loop binding protein mRNA processing, histone mRNA 3'-end
processing 206333_at MSI1 musashi homolog 1 (Drosophila) nervous
system development 206770_s_at SLC35A3 solute carrier family 35
(UDP-N- UDP-N-acetylglucosamine metabolic acetylglucosamine
(UDP-GlcNAc) process, transport, transporter), member A3 209307_at
SWAP70 SWAP-70 protein somatic cell DNA recombination, isotype
switching 211089_s_at NEK3 NIMA (never in mitosis gene a)-related
protein amino acid phosphorylation kinase 3 mitosis 211341_at
LOC100131317, POU4F1 similar to hCG1781072, POU class 4
transcription, regulation of transcription, homeobox 1
DNA-dependent, regulation of transcription from RNA polymerase II
promoter 212511_at PICALM phosphatidylinositol binding clathrin
protein complex assembly, endocytosis, assembly protein
receptor-mediated endocytosis 212830_at MEGF9 multiple
EGF-like-domains 9 NA 212999_x_at hCG_1998957, HLA- major
histocompatibility complex, class II, antigen processing and
presentation of DQB1/2, HLA-DRB1/2/ DR beta 1/2/3/4/5; similar to
major peptide or polysaccharide antigen via 3/4/5
histocompatibility complex, class II, DQ MHC class II beta 1
213501_at ACOX1 acyl-Coenzyme A oxidase 1, palmitoyl generation of
precursor metabolites and energy, lipid metabolic process 213831_at
HLA-DQA1 major histocompatibility complex, class II, antigen
processing and presentation of DQ alpha 1 peptide or polysaccharide
antigen via MHC class II 217954_at NA NA NA 217182_at MUC5AC mucin
5AC, oligomeric mucus/gel-forming cell adhesion, digestion, fibril
organization and biogenesis 217322_x_at NA NA NA 217777_s_at
PTPLAD1 protein tyrosine phosphatase-like A domain I-kappaB
kinase/NF-kappaB cascade containing 1 218803_at CHFR checkpoint
with forkhead and ring finger protein polyubiquitination, mitotic
cell domains cycle, ubiquitin-dependent protein catabolic process
219425_at SULT4A1 sulfotransferase family 4A, member 1 lipid
metabolic process, steroid metabolic process 221663_x_at HRH3
histamine receptor H3 signal transduction, G-protein coupled
receptor protein signaling pathway, neurotransmitter secretion
223977_at TMOD3 tropomodulin 3 (ubiquitous) NA 224327_s_at DGAT2
diacylglycerol O-acyltransferase homolog 2 glycerol metabolic
process, lipid (mouse) metabolic process, lipid biosynthetic
process, triacylglycerol biosynthetic process 224996_at NA NA NA
225579_at PQLC2 PQ loop repeat containing 3 NA 226240_at MGC21874
transcriptional adaptor 2 (ADA2 homolog, transcription, regulation
of transcription, yeast)-beta DNA-dependent 227280_s_at CCNYL1
Cyclin Y-like 1 NA 227618_at NA NA NA 227983_at RILPL2 Rab
interacting lysosomal protein-like 2 NA 228980_at RFFL ring finger
and FYVE-like domain intracellular protein transport, apoptosis
containing 1 229191_at TBCD tubulin folding cofactor D protein
folding, beta-tubulin folding 230836_at ST8SIA4 ST8
alpha-N-acetyl-neuraminide alpha-2,8- protein modification process
protein sialyltransferase 4 amino acid glycosylation, nervous
system development 231599_x_at DPF1 D4, zinc and double PHD fingers
family 1 transcription, regulation of transcription, DNA-dependent,
induction of apoptosis 234495_at KLK15 kallikrein-related peptidase
15 proteolysis 234986_at NA NA NA 234987_at NA NA NA 236232_at STX4
Syntaxin 4 transport, neurotransmitter transport, intracellular
protein transport 236404_at NA NA NA 236698_at NA NA NA 238327_at
LOC440836 similar to MGC52879 protein cell growth 238445_x_at
MGAT5B mannosyl (alpha-1,6-)-glycoprotein beta- NA
1,6-N-acetyl-glucoaminyltransferase, isozyme B 239463_at NA NA NA
242383_at NA NA NA 242563_at NA NA NA 243819_at NA NA NA 244841_at
SEC24A SEC24 related gene family, member A (S. cerevisiae)
transport, intracellular protein transport, ER to Golgi
vesicle-mediated transport 32069_at N4BP1 NEDD4 binding protein 1
NA 44673_at SIGLEC1 sialic acid binding Ig-like lectin 1,
inflammatory response, cell adhesion sialoadhesin 53720_at C19orf66
chromosome 19 open reading frame 66 NA
TABLE-US-00015 TABLE 15 Most predictive gene signatures identified
by MiPP in a dataset of patients with myocarditis (n = 16) vs
idiopathic dilated cardiomyopathy in training (n = 32): Validation
was performed in independent test sets (n = 18). Selection Class
Mean ER in Mean ER in Gene signatures method Prediction rule
comparison training set validation set MSI1, 1556507_at MiPP
SVM-rbf 2 0 0.167 KRT78 MiPP SVM-lin 2 0.033 0.167 KRT78,
1556507_at MiPP QDA 2 0 0.167 KRT78, 1556507_at MiPP LDA 2 0 0.167
1556507_at MiPP LDA, QDA, SVM-rbf 2 0 0.167
TABLE-US-00016 TABLE 16 Models obtained from 50 random splits into
train and test set: Genes obtained from 50 random splits were
further validated in 200 independent random splits. Illustrated are
the results from the top 5 gene clusters with the lowest mean error
(ER). Mean sMipp is an additional parameter for performance and
converges towards 1, as accuracy of the model increases. mean mean
5% 50% 95% Split Gene1 Gene2 Gene3 Gene4 Gene5 Gene6 ER sMiPP ER ER
ER 17 KRT78 1556507_at NA NA NA NA 0.078 0.789 0.188 0.063 0 45
KRT78 1556507_at NA NA NA NA 0.078 0.789 0.188 0.063 0 44 MSI1
POU4F1 1556507_at NA NA NA 0.09 0.776 0.188 0.063 0 43 MSI1 POU4F1
1556507_at LCE1E NA NA 0.091 0.789 0.188 0.063 0 41 LCE1E POU4F1
MSI1 NA NA NA 0.092 0.791 0.188 0.063 0
TABLE-US-00017 TABLE 17 Realtime RT-PCR data of patients with
lymphocytic myocarditis (n = 10) vs idiopathic dilated
cardiomyopathy (n = 10). Fold Fold Change Change P < 0.05 P <
0.05 Gene by by by by Probe Set Symbol SAM qPCR SAM qPCR
201721_s_at CD14 +5.9 +6.8 Y Y 1554899_s_at FCER1G +5.3 +5 Y Y
210146_x_at TLR1 +4.5 +4.2 Y Y 204923_at TLR2 +3.9 +5.9 Y Y
1555349_a_at ITGB2 +3.1 +1.95 Y Y 44673_at SIGLEC1 +2.3 +4.3 Y Y
219938_s_at TLR7 +2.3 +2.8 Y Y 203741_s_at ADCY7 +2 +4.2 Y Y
212830_at MEGF9 +1.5 +2.3 Y Y 217777_s_at PTPLAD1 +1.5 +1.7 Y Y
209307_at SWAP70 +1.4 +2.1 Y Y 206333_at MSI1 -1.8 -8.4 Y Y
1559224_at LCE1E -2.3 -2.6 Y Y
TABLE-US-00018 TABLE 18 Identification of subtypes of inflammatory
cardiomyopathy vs IDCM. Transriptomic Overall biomarker Sensitivity
Specificity PPV NPV accuracy Subtype (number of genes) (%, 95CI)
(%, 95CI) (%, 95CI) (%, 95CI) (%) Giant cell 8 67 (13-98) 92
(62-100) 67 (13-98) 92 (62-100) 86 myocarditis Sarcoidosis 58 89
(51-99) 67 (35-89) 67 (35-89) 89 (51-99) 77 Peripartum 56 83
(36-99) 67 (35-89) 56 (23-85) 89 (51-99) 74 cardiomyopathy Systemic
lupus 21 50 (9-91) 100 (71-100) 100 (20-100) 87 (58-98) 76
erythematosus
TABLE-US-00019 TABLE 19 Classifier to distinguish rare subtypes of
inflammatory cardiomyopathy from lymphocytic myocarditis.
Transriptomic Overall biomarker Sensitivity Specificity PPV NPV
accuracy Subtype (number of genes) (%, 95CI) (%, 95CI) (%, 95CI)
(%, 95CI) (%) Giant cell 4 100 (31-100) 100 (82-100) 100 (31-100)
100 (82-100) 100 myocarditis Sarcoidosis 6 100 (63-100) 100
(82-100) 100 (63-100) 100 (82-100) 100 Peripartum 12 100 (52-100)
100 (82-100) 100 (52-100) 100 (82-100) 100 cardiomyopathy Systemic
lupus 27 25 (1-78) 91 (70-98) 33 (2-87) 88 (67-97) 81
erythematosus
[0174] Although the invention has been illustrated and described
with respect to one or more implementations, equivalent alterations
and modifications will occur to others skilled in the art upon the
reading and understanding of this specification and the annexed
drawings. In addition, while a particular feature of the invention
may have been disclosed with respect to only one of several
implementations, such feature may be combined with one or more
other features of the other implementations as may be desired and
advantageous for any given or particular application.
[0175] The Abstract of the Disclosure is provide to allow the
reader to quickly ascertain the nature of the technical disclosure.
It is submitted with the understanding that it will not be used to
interpret or limit the scope or meaning of the following
claims.
* * * * *