In this study, we selected 25 Chinese Han pregnancies clinically diagnosed with VTE, and 17 normal controls and performed WES to identify pathogenic variants. WES analysis revealed 5810 variants of 3417 genes in unrelated subjects. 56 variants annotated in 46 genes were significantly different between maternal VTE cases and controls (P < 0.001). Furthermore, 306 variants that were absent in controls were obtained. Moreover, some variants were selected by MAF. By the logistic regression of 70 selected variants, 6 variants including rs7099478 in GRK5, rs8041208 in WDR72, rs17215792, rs13035688, rs6725221 and rs3214417 in KLF7, were associated with an increased risk of VTE in pregnant patients (P < 0.05, OR > 1).
Moreover, the biological processes showed that these significantly differentiated genes were strongly related to DNA replication initiation. The molecular function clusters showed that these genes were strongly related to functions involving ions. The cellular component enriched these genes in the perikarya and mitochondria. The most highly enriched class of pathway was for the activation of the pre-replicative complex (P = 0.058) with 2 genes including CDC7 and MCM6. CDC7 (cell division cycle 7) encodes a cell division cycle protein with kinase activity that is critical for the G1/S transition. MCM6 (Mini-chromosome maintenance complex component 6) encodes a putative replicative helicase. There has been Activation of ATR in response to replication stress with the same 2 genes identified in our study. A total of 5 direct connections among 7 genes (KCNC2, CCK, GRK5, ABCA4, SEMA4A, CDC7, MCM6) were observed by PPI analysis. We presumed that these variants may contribute to protein function by involving DNA replication and ion mediated molecular signaling, which underlies VTE. Notably, GRK5 (G protein-coupled receptor kinase 5) encodes a member of the guanine nucleotide-binding protein (G protein)-coupled receptor kinase subfamily of the Ser/Thr protein kinase family and plays a role in regulating the motility of polymorphonuclear leukocytes (PMNs). GRK5 was also found to be significantly related to VTE in pregnancy by logistic regression. The other extensive network was consistent with the pathway results including CDC7 and MCM6, both of which are related to cell division and DNA replication in tissue. In summary, it has been suggested that GRK5, CDC7, and MCM6 may have potential functions involving VTE in pregnancies.
Several well-studied variants were detected, including F5, F2, MTHFR, ACE, FGG, FGB, TES, PTS, PTH, PROC, and PROS1. However, the results showed that the differences in these factors between cases and controls were not statistically significant.
To date, there have been no studies on relevant maternal VTE genetic factors focusing on populations in mainland China. In our study, 6 variants were identified by the WES and logistic regression analyses of selected variants for VTE in pregnancy. The OR value were > 2.0, which may be an important reference for thrombosis causing VTE in pregnancies. Most previous genetic studies have found that variations for statistically significant genes show a small odds ratio to disease (usually less than 1.2). However, the 95% CI range for each site was larger in our study because of the small sample size. Another limitation is that the GO and PPI analyses involved only small number of genes, which may miss some important genetic factors.
The common risk factors of VTE include age, pregnancy, delivery, oral contraceptives, hormone replacement therapy (HRT), antiphospholipid antibody syndrome, trauma, surgery, malignant tumor, diabetes, nephrotic syndrome, long-distance travel, and long-term bed rest. In addition, several genetic factors, including PC, PS, PT gene mutation G20210A, FVL F5, and AT were observed . Previous VTE case-control studies of Chinese pregnant women regarding risk factors for VTE showed no significance. To date, a study in Hong Kong (case: control = 44:55) showed that no significance of factor V Leiden and G20210A mutation was identified. Furthermore, a deficiency in the antithrombin gene was observed in only 5 cases (11%) . To date, the largest survey of anticoagulants in the healthy population (n = 3493) in China  found that the detection rates of PC, AT, and PS deficiency were 0.29%, 0.08%, and 0.056%, respectively, and the gene variation rate was 0.43%. Another study showed  no mutation of factor V Leiden and prothrombin G20210A in VTE patients, PE patients, and normal controls. These results were consistent with our analysis. Further studies with large sample sizes are needed.
Notably, KLF7, with 4 variants present in 14 cases, was strongly associated with VTE in pregnancies. The rs17215792-AC, rs13035688-TA, rs6725221-CG and rs3214417-CA/C genotypes (OR > 1) were associated with increased risk of VTE in pregnant patients. It suggested that KLF7 may have an impact on VTE pathogenesis.
The KLF7 (Krüppel-like factor 7) gene is located on human chromosome 2q33.3and contains 11 exons according to the NCBI database. The KLF7 protein belongs to the Krüppel-like transcriptional regulator family, also known as ubiquitous Krüppel-like factor (UKLF), which plays an important role in regulating cell proliferation, differentiation, and survival. The KLF7 protein contains three C2H2 zinc fingers at the C-terminus that mediate binding to guanine-cytosine rich sites. The KLF7 protein is ubiquitously expressed in the bone marrow, endometrium, fat, and other various tissues.
In 1998, KLF7 was initially cloned from human vascular endothelial cells by PCR. According to its wide expression in various adult tissues, it was named the ubiquitous KLF by Matsumoto .
Many factors of the Krüppel family (KLFs) participate in the physiological regulation of muscle cells. KLF7 plays an important role in regulating nerve development and myeloid cell proliferation and is expressed in skeletal muscles and myoblasts. A study found that KLF7 may affect the myoblast cell cycle.
In addition, variants of KLF7 have been previously reported to cause obesity and type 2 diabetes, involving fat cell differentiation [24, 25]. It was reported that KLF7 can regulate a number of genes related to fat synthesis and metabolism, including lipoprotein lipase (LPL) and leptin (LEP), which are known to be associated with VTE [26, 27].
KLF7 is also involved in coronary artery disease (CAD) . KLF7 may bind to the promoter region of CAD-associated genes (factor VII, platelet derived growth factor, leptin, and plasminogen) as shown by bioinformatics prediction. Moreover, KLF7 may play important roles in ischemic heart disease (IHD) with hypercholesterolemia, arterial hypertension, and type 2 diabetes .
These findings indicate that lipid metabolism genes, such as KLF7, may be involved in the formation of cardiovascular continuum comorbidity by regulating glucose and lipid metabolism.
In our study, 4 variants in KLF7 were all located in the 3’ UTR region. This indicates that these mutations may alter KLF7 expression by regulating its binding to the miRNA of fat or muscle function genes.
Therefore, further in-depth research is needed to explore how the KLF7 gene affects cardiovascular disease, as well as VTE, obesity, and glucose dysfunction disorders.