3.1 Pearson’s Correlation Testing and PCA
Pearson’s correlation test showed strong correlations between VTE and control samples in the GSE48000 dataset (Fig. 1A). The PCA profile for the GSE48000 data revealed that the distances between samples were small in the VTE groups and control groups, respectively (Fig. 1B). Pearson’s correlation analysis also indicated strong correlations for the GSE19151 data among the samples in the VTE group and control group, respectively (Fig. 1C). The close distance in the dimension of PCA illustrated the acceptable data repeatability between samples in the VTE group and control group for the GSE19151 dataset (Fig. 1D).
3.2 Identification of DEGs in VTE
As shown in Fig. 2, a total of 232 genes were designated as DEGs in the VTE group when compared with the control group. The volcano plots in this figure present the DEGs with a cutoff criteria of having an adjusted P-value < 0.05 and |log2fold change|>1 in the GSE48000 and GSE19151 datasets (Fig. 2A and 2B). As examples of these differences, the top 10 differentially expressed genes are reported in Table 1.
Table 1
Top 10 most upregulated DEGs and top 10 most downregulated DEGs in VTE
Gene name
|
Log2 fold change
|
Adjusted P-value
|
Gene Expression
|
EVI2A
|
2.15595960
|
2.46×10− 13
|
Upregulation
|
RPL9
|
1.89195402
|
1.48×10− 18
|
Upregulation
|
IFI27
|
1.87618320
|
1.88×10− 5
|
Upregulation
|
RPL31
|
1.77436415
|
3.44×10− 14
|
Upregulation
|
NDUFA4
|
1.74588189
|
7.28×10− 17
|
Upregulation
|
IGFBP1
SNORD8
RPS7
XK
RPS15A
FOS
JMJD1C
|
1.74512022
1.71070142
1.68354151
1.63316845
1.51993276
−1.37065523
−1.30709903
|
1.33×10− 7
5.02×10− 5
2.35×10− 13
1.00×10− 11
1.20×10− 15
1.43×10− 7
3.65×10− 6
|
Upregulation
Upregulation
Upregulation
Upregulation
Upregulation
Downregulation
Downregulation
|
ZFP36L2
CD46
SNX10
UBXN4
DICER1
LSP1
TMEM259
DCK
|
−1.30546256
−1.16530934
−1.14759082
−1.14648762
−1.13523701
−1.10000543
−1.05424088
−1.04088634
|
9.36×10− 10
7.51×10− 6
2.10×10− 5
1.22×10− 5
3.46×10− 9
5.98×10− 24
4.66×10− 13
1.11×10− 7
|
Downregulation
Downregulation
Downregulation
Downregulation
Downregulation
Downregulation
Downregulation
Downregulation
|
3.3 Enrichment of DEGs by GO and KEGG Analysis
Gene functional enrichment analysis was performed to analyze the biological connections of these DEGs. Results of Gene Ontology (GO) enrichment analysis revealed that RNA catabolic process, viral gene expression, SRP-dependent cotranslational protein targeting to membrane, and viral transcription were the main biological processes (BPs) (Fig. 3A), and structural constituent of ribosome, cytochrome-c oxidase activity, and heme-copper terminal oxidase activity were the most enriched categories of molecular functions for these DEGs (Fig. 3B). The variations in cell component (CC) of DEGs were enriched largely in ribosome and hemoglobin complex (Fig. 3C). KEGG pathway analysis indicated that these DEGs were mainly involved in particular pathways, such as the ribosome, Huntington disease, and oxidative phosphorylation (Fig. 3D). Metascape was used to visualize these gene enrichment analyses to verify our results from R (Fig. 3E). We found that these DEGs were enriched in amino acid deficiency, ribosomal complex, oxidative phosphorylation, rRNA transcript, and blood coagulation.
3.4 Identification of Probable Disease-causing DEGs by WES
WES revealed 48 mutations of DEGs in the VTE group. The mutation types and the log2fold change are shown in Table 2. Because nonsynonymous mutations are most likely to affect protein function, we focused on the four SNP variants corresponding with four DEGs in the VTE group. These were HNMT (ch2: 138759649 C > T, rs11558538, adjusted P-value = 1.2 × 10− 9), POLL (chr10: 103340056 G > A, rs3730477, adjusted P-value = 5.12 × 10− 4), ZNF292 (chr6: 87925827 A > G, rs9362415, adjusted P-value = 2.95 × 10− 8), and DPCD (chr10: 103361088 C > T, rs7874, adjusted P-value = 4.36 × 10− 5). The adjusted P-value of HNMT was the lowest in this study. Functional analysis showed that most disease-causing DEGs were involved in anemia, sickle cell, pulmonary thromboembolisms, heparin-induced thrombocytopenia, thrombophilia, and so on, as shown by Metascape functional analysis (Fig. 4). As summarized in Table 3, we found that HNMT was expressed in heparin-induced thrombocytopenia, dermatitis, and atopic cases, conditions that may have strong impacts on VTE.
Table 2
Probable disease-causing DEGs of VTE
Gene
|
Chr
|
SNP
|
Mut_type
|
Mutation
|
Location
|
Position
|
Func.ref
|
Exonicfunc.ref
|
1 KG
|
ExAC_ALL
|
Cases(n = 25) Controls(n = 17) logFC Adj.P.Val
|
HNMT
|
ch2
|
rs11558538
|
SNP
|
C/T
|
ontarget
|
138759649
|
exonic
|
nonsynonymous SNV
|
0.0595048
|
0.1017
|
1
|
5
|
1.051877
|
1.2×10− 9
|
USP14
|
chr18
|
rs56806027
|
SNP
|
T/A
|
flank150
|
204815
|
intronic
|
|
0.68111
|
|
2
|
7
|
1.265047
|
0.0001484
|
|
|
rs57035428
|
InDel
|
T/TAAAAA
|
flank150
|
204816
|
intronic
|
|
|
|
2
|
7
|
|
|
SERPING1
|
chr18
|
rs17072114
|
SNP
|
T/C
|
flank150
|
61584817
|
intronic
|
|
0.171326
|
|
5
|
9
|
1.0924736
|
0.0005806
|
UBXN4
|
chr2
|
rs80198954
|
SNP
|
C/A
|
ontarget
|
136511842
|
exonic
|
synonymous SNV
|
0.0373403
|
0.019
|
6
|
12
|
-1.146488
|
0.0003692
|
|
|
rs74265494
|
SNP
|
G/A
|
ontarget
|
136511886
|
intronic
|
|
0.0397364
|
0.0191
|
6
|
12
|
|
|
|
|
rs372143998
|
InDel
|
GT/G
|
flank150
|
136527319
|
intronic
|
|
|
0.2468
|
6
|
11
|
|
|
|
|
rs200613240
|
InDel
|
T/TA
|
flank150
|
136529897
|
intronic
|
|
|
|
0
|
9
|
|
|
|
|
rs78878675
|
SNP
|
G/A
|
ontarget
|
136530157
|
intronic
|
|
0.0397364
|
0.019
|
6
|
12
|
|
|
|
|
rs78339162
|
SNP
|
A/G
|
flank150
|
136533993
|
intronic
|
|
0.0397364
|
|
6
|
11
|
|
|
ZNF271P
|
chr18
|
rs12965288
|
SNP
|
C/A
|
ontarget
|
32888090
|
ncRNA_exonic
|
|
0.249401
|
|
2
|
7
|
1.0913303
|
1.0913303
|
|
|
rs34841246
|
SNP
|
C/A
|
ontarget
|
32888546
|
ncRNA_exonic
|
|
0.220847
|
|
3
|
8
|
|
|
SELP
|
chr1
|
rs35706397
|
InDel
|
T/TA
|
ontarget
|
169560727
|
intronic
|
|
0.268371
|
0.3319
|
8
|
21
|
1.0297047
|
0.0002834
|
GYPA
|
chr4
|
rs62334651
|
SNP
|
T/C
|
flank150
|
145040784
|
intronic
|
|
0.402556
|
|
12
|
2
|
1.0422875
|
1.19×10− 08
|
|
|
rs62334653
|
SNP
|
G/A
|
flank151
|
145041036
|
intronic
|
|
|
|
11
|
3
|
|
|
TMEM259
|
chr19
|
rs2240161
|
SNP
|
A/G
|
ontarget
|
1011823
|
intronic
|
|
0.674321
|
0.7045
|
14
|
15
|
-1.054241
|
1.52×10− 11
|
|
|
rs7146
|
SNP
|
A/G
|
ontarget
|
1014398
|
exonic
|
synonymous SNV
|
0.67512
|
0.6579
|
14
|
15
|
|
|
POLL
|
chr10
|
rs1055364
|
SNP
|
C/A
|
ontarget
|
103338730
|
UTR3
|
|
0.163339
|
|
0
|
4
|
1.1418465
|
0.0005122
|
|
|
rs1055362
|
SNP
|
A/G
|
ontarget
|
103338733
|
UTR3
|
|
0.163339
|
|
0
|
4
|
|
|
|
|
rs3730477
|
SNP
|
G/A
|
ontarget
|
103340056
|
exonic
|
nonsynonymous SNV
|
0.0998403
|
0.1678
|
0
|
4
|
|
|
|
|
rs3730476
|
SNP
|
A/G
|
ontarget
|
103340144
|
exonic
|
synonymous SNV
|
0.163139
|
0.2306
|
0
|
4
|
|
|
|
|
rs3730475
|
SNP
|
A/G
|
ontarget
|
103340179
|
intronic
|
|
0.163339
|
0.232
|
0
|
4
|
|
|
|
|
rs3730474
|
SNP
|
T/C
|
flank150
|
103340235
|
intronic
|
|
0.163139
|
|
0
|
4
|
|
|
|
|
rs3730465
|
SNP
|
A/G
|
flank150
|
103343533
|
intronic
|
|
0.164936
|
|
0
|
4
|
|
|
|
|
rs3730462
|
InDel
|
CTGTTG/C
|
ontarget
|
103345941
|
intronic
|
|
0.162939
|
0.2281
|
0
|
4
|
|
|
ASTN1
|
chr1
|
rs868002876
|
InDel
|
AT/A
|
ontarget
|
176913216
|
intronic
|
|
0.431909
|
|
1
|
5
|
1.0697509
|
0.0010276
|
UGGT1
|
chr2
|
rs35069237
|
InDel
|
GT/G
|
ontarget
|
128949841
|
UTR3
|
|
0.338858
|
|
7
|
12
|
1.1432353
|
0.0007117
|
NFATC1
|
chr18
|
rs8096658
|
SNP
|
C/G
|
flank150
|
77156537
|
intronic
|
|
0.328275
|
|
0
|
4
|
1.0968715
|
0.0000459
|
|
|
rs56376587
|
SNP
|
A/C
|
flank150
|
77160235
|
intronic
|
|
0.332268
|
|
3
|
8
|
|
|
MTHFR
|
chr1
|
rs11121832
|
SNP
|
T/C
|
flank150
|
11860120
|
intronic
|
|
0.759185
|
|
18
|
17
|
1.1058389
|
0.0001734
|
FCGR1B
|
chr1
|
rs827371
|
SNP
|
T/C
|
flank150
|
120935661
|
intronic
|
|
|
|
4
|
8
|
1.0236005
|
2.69×10− 11
|
MAP3K8
|
chr10
|
rs3034
|
SNP
|
G/A
|
flank150
|
30749895
|
UTR3
|
|
0.882987
|
|
21
|
9
|
-1.01569
|
0.0002499
|
MGMT
|
chr10
|
rs2782888
|
SNP
|
T/G
|
flank150
|
131265328
|
upstream
|
|
1
|
|
18
|
17
|
1.0357623
|
0.0001873
|
|
|
rs55973415
|
SNP
|
G/A
|
flank150
|
131557750
|
intronic
|
|
0.2143
|
|
6
|
10
|
|
|
ZNF2929
|
chr6
|
rs563101504
|
InDel
|
GACACAC/G
|
ontarget
|
87925827
|
intronic
|
|
|
|
1
|
5
|
1.0186153
|
2.95×10− 08
|
|
|
rs9362415
|
SNP
|
A/G
|
ontarget
|
87968565
|
exonic
|
nonsynonymous SNV
|
0.592851
|
0.5996
|
24
|
12
|
|
|
|
|
rs3734187
|
SNP
|
C/T
|
ontarget
|
87969737
|
exonic
|
synonymous SNV
|
0.592652
|
0.5876
|
24
|
12
|
|
|
|
|
rs3812132
|
SNP
|
C/G
|
ontarget
|
87969737
|
exonic
|
synonymous SNV
|
0.674121
|
0.5867
|
24
|
12
|
|
|
|
|
rs35541349
|
InDel
|
G/GA
|
flank150
|
87969737
|
UTR3
|
|
|
|
12
|
1
|
|
|
FOS
|
chr14
|
rs1063169
|
SNP
|
G/T
|
flank150
|
75747118
|
intronic
|
|
0.113019
|
|
8
|
0
|
-1.370655
|
0.0000396
|
ZNF346
|
chr5
|
rs11448853
|
InDel
|
A/AG
|
flank150
|
176471286
|
intronic
|
|
0.4742
|
|
11
|
14
|
1.057501
|
0.0001975
|
ERF
|
chr19
|
rs61735151
|
SNP
|
G/A
|
ontarget
|
42753283
|
exonic
|
synonymous SNV
|
0.0778754
|
0.0878
|
25
|
13
|
1.0310346
|
0.0004987
|
ALKBH89
|
chr11
|
rs589316
|
SNP
|
G/A
|
flank150
|
107402887
|
intronic
|
|
0.35603
|
|
3
|
9
|
1.034199
|
0.0035681
|
|
|
rs71488261
|
SNP
|
T/A
|
flank150
|
107422440
|
intronic
|
|
0.261981
|
|
0
|
4
|
|
|
WDR55
|
chr5
|
rs2251860
|
SNP
|
T/C
|
ontarget
|
140048209
|
exonic
|
synonymous SNV
|
0.508986
|
0.4722
|
16
|
16
|
1.1114745
|
0.0003608
|
AHSP
|
chr16
|
rs10843
|
SNP
|
T/C
|
ontarget
|
31540030
|
UTR3
|
|
0.0946486
|
0.1185
|
3
|
8
|
1.2937014
|
4.95×10− 11
|
DPCD
|
chr10
|
rs7911520
|
SNP
|
A/G
|
flank150
|
103354554
|
intronic
|
|
0.169329
|
|
0
|
4
|
1.4046498
|
0.0000436
|
|
|
rs7874
|
SNP
|
C/T
|
ontarget
|
103361088
|
exonic
|
nonsynonymous SNV
|
0.0998403
|
0.168
|
0
|
4
|
|
|
Table 3
Functional enrichment analysis of disease-causing DEGs using Metascape
GO
|
Description Log10P Count Genes
|
C0002895
|
Anemia Sickle cell
|
−5.9
|
6
|
FOS|GYPA|MTHFR|SELP|AHSP|UGGT1
|
C0524702
|
Pulmonary thromboembolisms
|
−5.5
|
3
|
MTHFR|SELP|USP14
|
C0002875
|
Cooley's anemia
|
−5.2
|
4
|
GYPA|MTHFR|AHSP|UGGT1
|
C0272285
|
Heparin-induced thrombocytopenia
|
−5.1
|
3
|
FCGR1B|HNMT|SELP
|
C0004135
|
Ataxia telangiectasia
|
−4.8
|
5
|
FOS|GYPA|MGMT|MTHFR|NFATC1
|
C0268138
|
Xeroderma pigmentosum
|
−4.5
|
3
|
MGMT|MTHFR|UGGT1
|
C0011615
|
Dermatitis, Atopic
|
−4.5
|
6
|
ASTN1|FOS|HNMT|MGMT|MTHFR|SELP
|
C0008626
|
Congenital-chromosomal disease
|
−4.5
|
6
|
FCGR1B|FOS|MGMT|MTHFR|NFATC1|SELP
|
C0278996
|
Malignant chromosomal disease
|
−4.4
|
6
|
FCGR1B|FOS|MGMT|MTHFR|NFATC1|SELP
|
C3887461
|
Head and neck carcinoma
|
−4.4
|
6
|
FCGR1B|FOS|MGMT|MTHFR|NFATC1|SELP
|
C0014170
|
Endometrial neoplasms
|
−4.2
|
4
|
MAP3K8|FOS|MGMT|MTHFR
|
C0947751
|
Vascular inflammations
|
−3.9
|
4
|
SERPING1|MAP3K8|FOS|SELP
|
C1704436
|
Peripheral arterial diseases
|
−3.9
|
4
|
MAP3K8|FOS|MTHFR|SELP
|
C0011884
|
Diabetic retinopathy
|
−3.7
|
5
|
SERPING1|MAP3K8|FOS|SELP|MTHFR
|
C0024814
|
Marinesco-Sjogren syndrome
|
−3.7
|
3
|
MAP3K8|MGMT|MTHFR
|
C0333516
|
Tumor necrosis
|
−3.7
|
4
|
FOS|MGMT|MTHFR|SELP
|
C3469521
|
Fanconi anemia
|
−3.6
|
4
|
GYPA|MGMT|MTHFR|SELP
|
C4551686
|
Malignant neoplasm of soft tissue
|
−3.6
|
5
|
MAP3K8|FOS|MGMT|MTHFR|NFATC1
|
C0015625
|
Fanconi anemia
|
−3.5
|
4
|
GYPA|MGMT|MTHFR|SELP
|
C0398623
|
Thrombophilia
|
−3.5
|
3
|
SERPING1|MTHFR|SELP
|
3.5 Protein Structure and Characterization of Missense HNMT Mutations
The Thr105Ile (rs11558538) polymorphism in the HNMT gene (ch2: 138759649 C > T, rs11558538, adjusted P-value = 1.2 × 10− 9) was the biggest difference identified in a gene, and should result in nonsense-mediated decay and loss function of this protein. The 3D location is shown in Fig. 5A and Fig. 5B. The variant was positioned in the α-helix, where its side chain hydroxyl formed two hydrogen bonds with a backbone oxygen after mutation, causing a marked decrease in the levels of both HNMT enzymatic activity and immunoreactive protein[26, 27]. HNMT is an enzyme that has been implicated in neurotransmission by inactivating histamine in the central nervous system[28]. However, histamine increases vascular permeability through the histamine H1 receptor to activate nerve endings, relaxing vascular smooth muscle[29].
3.6 Molecular Docking
The drug–target interactions for HNMT were predicted using DGIdb, and the results are presented in Table 4, providing a theoretical therapeutic mechanism for VTE prevention. Six drugs targeting HNMT have been predicted for VTE, including Amodiaquine, Harmaline, Aspirin, Metoprine, Dabigatran, and Diphenhydramine.
Molecular docking analysis was attempted to assess the potential noncovalent binding of HNMT with these small molecules drugs. In general, a lower binding energy indicated a stronger binding between HNMT and a compound. Table 4 shows the six drugs that best interfaced with HNMT. To visualize these docking results, the 3D interaction diagrams of HNMT and their corresponding best-matched drugs were drawn, as shown in Fig. 6. These drugs, such as Aspirin and Dabigatran, have been utilized to recanalize vessels and prevent thrombi growth clinically in VTE patients[30–32]. The 3D interaction diagram of Aspirin at the active site of HNMT revealed that this interaction was stable through forming hydrogen bonds with the key residues Lys55 and Lys135 (Fig. 6B). Aspirin is commonly administered to inhibit platelet aggregation and prevent thrombus formation[33]. Additionally, three hydrogen bonds formed with residues Phe9, Tyr15, and Ser91 contributed to stabilizing the interaction between Dabigatran and HNMT. Dabigatran has been approved for use in orthopedic surgery, venous thromboprophylaxis, acute VTE treatment, and extended prevention of recurrent VTE[34]. Our data had shown that HNMT can potentially become a new target for VTE treatment.
Table 4
Candidate drugs targeted HNMT
Gene
|
Drug
|
Sources
|
PMIDs
|
Binding Energy (kcal. mol-)
|
Binding Residues
|
HNMT
|
Amodiaquine
|
DrugBank;
|
6789797
|
-2.48
|
GLN197;
ASP203
|
HNMT
|
Aspirin
|
DrugBank
|
19178400
|
-3.24
|
LYS55;
LYS135
|
HNMT
|
Harmaline
|
PharmGKB
|
1530666
|
-5.07
|
GLU28
|
HNMT
|
Metoprine
|
TTD;DTC
|
10592235
|
-3.65
|
GLN192;
ASP194
|
HNMT
|
Dabigatran
|
TTD
|
-
|
-4
|
PHE9;TYR15;SER91
|
HNMT
|
Diphenhydramine
|
DrugBank
|
23896426
|
-4.69
|
ASP194
|