Antiphospholipid syndrome (APS), previously termed as Hughes syndrome, is a chronic autoimmune disease characterized with venous or arterial vascular thrombosis and pregnancy morbidity (1). Reliable epidemiological studies on antiphospholipid antibody (aPL) and APS are still largely lacking. Limited statistics just show that the annual incidence of APS is about 2-5 cases per 100,000, the prevalence is about 40-50 cases per 100,000 and the 10-year mortality rate is approximately 10% (2–4). Currently, the key laboratory tests for clinical diagnosis of APS are three standard aPLs: anti-cardiolipin antibodies (aCL), anti-β2 glycoprotein I antibody (aβ2GPI) and lupus anticoagulant (LA). APS patients often have multiple organs-systems injury and develop genetical predisposition, associated with the persistent presence of serum aPL (5). However, there are many problems in the practical application of the above three standard aPLs. APS patients have diverse clinical manifestations, even some atypical clinical manifestations are also frequently seen in APS patients. Numerous scholars have proposed non-standard clinical presentations and called for the refinement of new criteria to classify patients with increasingly recognized non-standard neurological presentations (6, 7). The expression of aPLs is relatively complex, suggesting that there may be non-aPL related clinical manifestations (8). Some reserchers have proposed a new type of APS: seronegative APS (SN-APS) (9). In some cases, patients may present with clinical features of APS but with temporary positive or persistently negative titers of aPL, meaning that these patients with high clinical suspicion of APS but cannot be diagnosed as APS because they do not meet the laboratory test diagnosis criteria, which may cause a certain impact on the treatment and prognosis of patients. Moreover, Gilberto et al. have proposed “non-criteria” APS subgroups, such as aPL carriers and patients with “non-criteria” manifestations and positivity for “non-criteria” aPL (10). Although many studies have explored the clinical value of non-standard antiphospholipid antibodies, which may provide hints on some conditions (11, 12), the new APS classification criteria process is well under way and some initiatives are being proposed (13–15). It is reported the committee recommend the removal of tests for anti-domain-I antibodies (aDI), anti-phosphatidylserine-prothrombin antibodies (aPS/PT), aCL IgA, aβ2GPI IgA, anti-prothrombin antibodies, resulting from their limited commercial availability, additional studies which are required to define feasibility, clinical relevance and standardization. However, the positive rate of the same type of antibody varies greatly among different populations in many studies. In addition, aPL is also positively expressed in other autoimmune diseases, infections, tumors, vaccinations, and even healthy individuals (16). 30-50% of patients with systemic lupus erythematosus (SLE) are reported to be positive for at least one aPL test (17), meaning the specificity and sensitivity of aPLs may not meet the needs of clinical diagnosis and treatment. These problems together lead to the heterogeneity and complexity in the diagnosis and prognosis of APS patients, and bring a double burden to patients mentally and economically. Therefore, early diagnosis, early treatment and intervention, drug action prediction and prognosis of APS are important clinical problems to be solved urgently. Reserches with larger number of samples and longer follow-up time are still needed to judge the application value of non-standard antibodies in future.

In recent years, the continuous development and innovation of NGS (next-generation sequencing) technology has greatly promoted the integration and development of all fields of genome research (18). NGS technology mainly obtains DNA sequence data by library construction, cross-linking amplification between library and vector, synthesis and sequencing on top of vector (19). At present, this technology is gradually moving from the research field to clinical practice, and the main technology categories include: whole genome sequencing (WGS), whole exome sequencing (WES) and target region sequencing (TRS), etc (20). As an “open system” for exploring new information and identifying existing information, this technology have higher sequencing capacity, ability to multiplex samples, and lower sequencing cost if batched. The massive sequencing capacity of NGS permits its broad research and clinical applications in different fields, such as diagnosis of diseases (21), microbiome determanition (22), detection of gene mutations (23) and so on. By using new technologies such as NGS and omics, researchers have explored the pathogenesis, clinical phenotype and biomarkers of APS in different directions. Meanwhile, multiple international multidisciplinary research networks and cooperative projects have been established (24) to further promote the in-depth study of the core mechanism of APS pathogenesis, and promote the mining and verification of novel biomarkers. It provides a new way to improve the existing problems of early diagnosis and prognosis of APS.

The pathogenesis of APS is multifaceted, and a growing body of evidence supports that the development of APS is caused by aPL mediated, interactions between inflammatory and thrombotic mechanisms centered on disruption of coagulation and fibrinolysis, involving aPL-mediated activation of platelets, monocytes and endothelial cells, altered adhesion molecules and activation of pro-inflammatory cytokines, complement and neutrophil activation, and tissue factor upregulation and many others (3, 25, 26). However, the correlation between antibody titers and different disease phenotypes and disease severity, as well as the progression from asymptomatic patients to catastrophic anti-phospholipid syndrome (CAPS), still leaves a gap in research, so scholars have tried to investigate the nature of APS disease from a new perspective at the genetic level.

A number of studies first analyzed the genomic diversity of APS patients. Barinotti A et al. found two siblings with APS and carried out whole exome sequencing to analyze single nucleotide polymorphisms (SNPS), and found that there were many sites involved in hemostasis, immune response and protein coding of phospholipid metabolism. However, the correlation with APS is weak (27). In Meta-analysis, Islam et al. showed that 16 genes related to thrombolic APS (TAPS), and the proteins encoded by these genes were interrelated, mainly involved in two processes of coagulation cascade and immune response (28).

Some studies have also proposed that the expression of type-I interferons (IFN-1) gene is related to the pathogenesis of APS and other autoimmune diseases (29). In 2008, the results of a genome-wide array study showed that a total of 93 genes were differentially expressed between primary APS patients and healthy controls, with both type I and type II IFN-induced families showing upregulated expression in PAPS subjects (30). Perez-Sanchez et al. analyzed monocyte gene expression and found the similar differences in interferon signature genes in patients with PAPS and APS associated with SLE (31). Further transcriptome sequencing of neutrophils from primary APS patients and healthy controls revealed that IFIT1 (IFN-1 regulatory gene) was the most up-regulated gene, and IFIT3, IFI6, MX1 and HERC5 were also significantly increased in APS (32). These findings also suggest that changes in IFN gene expression may lead to activation of multiple pathogenic signaling pathways in APS.

In addition, complement system activation also plays a key role in aPLs-mediated pregnancy loss (33, 34). In the study of Chaturvedi et al, DNA exome capture sequencing was performed on CAPS patient samples, including 15 exons related to complement activation and regulation. The results showed that CAPS patients had a high rare germline variation rate (60%) in complement regulatory genes. The mutation rate (51.5%) was similar to that of atypical heomlytic uremic syndrome patients (35). However, this is still a controversial issue, as researchers have found high circulating levels of complement fission products in asymptomatic aPLs carriers (36). Samantha L Coss et al. (37) discuss the complex genetic polymorphisms in complement and their association with autoimmune diseases, potential mutations in these genes act as a strongly predisposing factor, leading to uncontrolled complement activation and a more severe thrombotic phenotype. Clearly, hemostasis and complement activation feature in the induction of thrombosis by aPLs. Since these enzymatic cascades are intrinsically linked, coagulation activation may be the cause and subsequent complement activation may be the consequence.

Recently, in a large whole-exome sequencing study of ethnic populations, variants with low allele counts were filtered and analyzed in combination with SNP frequency, amino acid substitution tolerance and mutation-carrying domain function, which revealed rare variants of P2RY8 allele were found in SLE and APS patients. Further investigation of the effect of P2RY8 variants on protein function indicates that the enhancement of P2RY8 signaling pathway may have the potential to prevent or treat systemic autoimmune diseases (38). The current diagnostic criteria for systemic autoimmune diseases are often ambiguous, ununiversal, or difficult to evaluate, which can lead to misdiagnosis or leave patients undiagnosed. However, there are widespread clinical and genetic similarities between rheumatic autoimmune diseases (39). The PRECISESADS project now has begun to reclassify major systemic autoimmune diseases based on underlying molecular mechanisms (40, 41).

These overlapping and complementary findings also prove that the clinical manifestations and disease course of APS are highly heterogeneous with many different pathogenesis. There are still many challenges in the study of its genetic background. However, the sequencing technology can further discover a variety of possible changes in the gene level of this disease, which is expected to integrate and verify various research data, discover new potential diagnostic markers and therapeutic targets in in-depth studies, provide evidence support for the development of detailed and personalized treatment strategies.

The etiology of APS is not well understood, and there are no effective measures to prevent APS. For patients without thrombotic events, especially women of childbearing age, the guidelines recommend thst lifestyle changes to prevent adverse events (74).

For the prognosis and management of APS patients, Amigo et al. (75) have developed a system to assess aPL-related organ damage in patients with APS. Experts in this field have developed several tools to assess the risk of recurrent thrombosis or pregnancy morbidity in APS based on the diverse characteristics of the disease population (76–82). Recent studies have shown that JAK2^V617F mutation can lead to an increased risk of thrombolism. For APS patients with JAK2^V617F mutation, thrombotic adverse events may occur at an earlier stage, and more aggressive antithrombotic therapy strategies are needed to treat and prevent recurrence (83). Moreover, genetic prethrombotic risk factors are well established in children with APS, with 45% of patients enrolled in the Ped-APS registry having more than one genetic prethrombotic risk factors. Pediatric APS patients often present with “non-classical” APL-related clinical findings that cannot be directly attributed to the thrombotic process, implying a clear distinction between SLE related and APL-related manifestations (56). Through a large range of long-term follow-up observation data, the prediction accuracy can be improved if related indexes such as genetics and genes can be added to the relevant prediction algorithm, but more verification is still needed.

In the past few years, many studies ( Table 1 ) have been conducted on genetic polymorphisms in APS patients, which provide references for the future use of molecular mapping to characterize patients. The application of NGS technology will obtain relatively complete characteristics and information of APS patients, including specific immune cell populations, molecular cloning and gene expression differences, etc. The data system is huge and complex. Clarifying effective parameter information closely related to the diagnosis of APS, thrombosis and pathological pregnancy is an important step in screening potential biomarkers specific for APS. Compared with clinical antibody detection, NGS method can greatly improve the sensitivity and specificity of detection. By increasing the sequencing depth, a large volume of differential information can be obtained, but at the same time, the detection cost is correspondingly increased. Therefore, for the application of NGS in the current post-clinical practice, relevant examination at the gene level should be mainly evaluated and validated in the APS population with certain thrombotic and obstetric manifestations, maybe gradually conducted for patients with no or low expression of aPLs antibody during the preliminary screening, but highly suspected of APS, to assist in early diagnosis and supplement the problem that APS diagnostic system dominated by standard aPLs cannot meet the needs of clinical diagnosis and treatment in the future.

It is expected that in the future, through clinical studies with a relatively large sample size, strict patient inclusion criteria will be set up. APS patients will be stratified according to clinical outcomes and aPLs expression profiles of patients, so as to obtain experimental data clearly related to APS clinical manifestations and aPLs expression while reducing the occurrence of analysis bias. We need to ensure the independence of the derived data as biomarkers, analyze the causes of various differences, enrich the pathogenesis of APS, and further confirm its value in the diagnosis and prognosis of APS. Further work should be focused on building on existing work to evaluate genetic risk factors that significantly contribute to thrombosis and adverse pregnancy events through multilevel and multi-dimensional bioinformatics analysis, and to better identify standard and non-standard clinical phenotypes, antiphospholipid antibody profiles, and their relevance to clinical outcomes, prognostic tools, and treatment strategies. Sequencing information can be combined with multiomics techniques, opening the door to the possibility of developing novel diagnostic criteria for APS and providing more options for targeted therapies to prevent clinical adverse events in APS.

QL designed and wrote the review, drew the tables. YT and SY wrote and revised this manuscript. LC revised this manuscript and reviewed the figures and tables. All authors gave final approval for publication.