Hypertension (HTN) is one of the leading modifiable risk factors for morbidity and mortality of cardiovascular disease (CVD) worldwide [1
]. Likewise, type 2 diabetes mellitus (T2DM) has become one of the global disease burdens [2
]. HTN and T2DM are closely interlinked due to similar risk factors including obesity, dyslipidemia, insulin resistance, endothelial dysfunction, vascular inflammation, atherosclerosis, and sequential inappropriate activation of renin-angiotensin system, etc. [3
]. Previous studies have shown that hypertension is at least twice as frequent in patients with diabetes compared with those who do not have diabetes [5
]. Accordingly, a considerable portion of the world population undergoes coexistent T2DM and HTN, which increases the risk of CVD and renal failure in individuals [6
]. Compared with the normotensive non-diabetic individuals, patients with HTN and T2DM comorbidity (HDC) have a four-fold increased risk of CVD [7
]. The number of patients diagnosed with HTN and T2DM comorbidity (HDC) is on the rise, resulting in increased medical expenses and resource utilization [8
]. Thus, further studies of biomarkers for monitoring and preventing the progression from HTN or T2DM towards HDC are timely and called for.
The glycan attachment to individual proteins is driven by specific enzymes and plays an indispensable role in almost all physiological processes [9
]. In general, N-glycome constructed in healthy individuals is fairly stable but can vary due to pathological conditions, especially during inflammation [10
]. Altered glycosylation pathways can have significant effects on protein structure and function that occur during the transition from healthy to diseased states. Due to the absence of the genetic template and the lack of stringency within the biosynthetic pathway, the glycan structures of individuals are influenced by the intricate interplay of genetic polymorphisms and environmental factors [11
]. Therefore, it is warranted to elucidate the role of altered glycome composition in the pathogenic immune response and to analyze the possibility of using N-glycosylation as a latent predictor for the development of multifactorial diseases, such as HDC.
As the most abundant antibody in human blood, immunoglobulin G (IgG) regulates systemic inflammation balance at multiple levels, and its function is largely influenced by glycosylation. It has been shown that the pro- and anti-inflammatory activities of IgG could no longer be demonstrated if N-glycans are completely removed [12
]. Aberrant IgG N-glycosylation has also been demonstrated to be involved in the pathogenesis such as aging [13
], HTN [14
], and T2DM [15
], which are relevant risk factors contributing to HDC. These observations make us speculate that changes of IgG N-glycosylation may be related to the pathogenesis of HDC by regulating inflammation. Understanding the underlying altered IgG N-glycosylation profiles that predispose individuals to HDC may help to develop novel protective immune-targeted and anti-inflammatory therapies and/or prevention approaches. However, the associations between IgG N-glycans and HDC have not been described in current literature.
There are 55 officially recognized ethnic minority groups within China in addition to Han majority (which constitute about 91.9% of the total population). The population size of Han Chinese and Chinese ethnic minorities are 1286.3 and 125.5 million, respectively (according to the seventh national census statistics of China conducted in 2020). The genetic background, culture, socioeconomic conditions, climate, geographical features, lifestyle, and dietary habit are substantially different between Han Chinese and other ethnic minorities. The Kirgiz (KGZ), Tajik (TJK), and Uygur (UIG) are three Chinese Muslim ethnic minorities, with a total population size of 11.94 million, inhabiting the Xinjiang Uygur Autonomous Region, the northwestern frontier of China, for generations, without intermarry with other ethnic groups [17
]. These Chinese Muslim minorities share relatively similar lifestyles, diet-related habits, living environment, culture, and genetic backgrounds, thereby are well-defined isolated populations with substantial ancestry contribution from Western Eurasian people [18
]. The prevalence of HTN and T2DM in the northwestern Chinese minority populations is on the rise and relatively higher than that in the Han Chinese, the majority ethnic group in China [20
]. Investigating the IgG N-glycan profiles of ethnic groups with different prevalence of HTN and T2DM could shed light on the potential population-specific and/or general IgG N-glycan patterns and their relevance to the pathogenesis of HDC.
In this study, we investigated the changes in IgG N-glycosylation profiles associated with HDC and their biomarker potential for distinguishing HDC from HTN, T2DM, and healthy individuals in the aforementioned populations. This work could further increase our understanding of HDC pathophysiology and may shed light on future studies investigating their potential for monitoring or preventing the progression from HTN or T2DM towards HDC.
To the best of our knowledge, this is the first investigation on the association and discriminative potential of IgG N-glycan traits in HDC versus HTN, T2DM, and healthy individuals in the pooling of three Chinese Muslim ethnic minorities (KGZ, TJK, and UIG), inhabiting the northwestern frontier of China, as well as the Han Chinese population.
HTN and T2DM are common comorbidities that are multifactorial diseases driven by many common pathogenic factors, including modifiable risk factors (obesity, dyslipidemia, insulin resistance, endothelial dysfunction, vascular inflammation, atherosclerosis, and sequential inappropriate activation of renin-angiotensin system, etc.) [3
], environmental factors, and genetic predisposition [4
]. IgG contains complex-type biantennary N-glycan structures, which activate or inhibit IgG Fcγ receptors (FcγRs) by altering the binding affinity of IgG, thereby decisively affecting effector functions of IgG [35
]. The pro- and anti-inflammatory actions of IgG are modulated by the specific N-glycan residues including galactose, fucose, sialic acid, and bisecting GlcNAc [9
]. Cumulative evidence indicates that the alteration of terminal modification in IgG N-glycan plays a vital role in the anti- or pro-inflammatory process. Previous studies reported that aberrant glycosylation of IgG (low levels of sialylation and core fucosylation, as well as the high level of bisecting GlcNAc) have been demonstrated to be involved in a range of specific health conditions related to HDC, including aging, obesity, HTN, T2DM, dyslipidemia, and metabolic syndrome [13
]. In the present study, the altered IgG N-glycosylation profile was also observed in the HDC patients, further suggesting that IgG N-glycome patterns are associated with the progression of inflammatory disorders, such as HDC, via inflammatory signaling. These findings present a situation where the role of IgG glycans in the pathogenesis of HDC extends beyond just a reflection of changes stemming from the onset of disease, but rather perhaps has some contribution towards influencing pathogenesis itself resulting from the altered inflammatory actions of IgG.
In this study, the higher level of GP5 (M5) and GP6 (FA2B), and the lower levels of GP16 (FA2G1S1) and GP18 (FA2G2S1) were found to be the population-specific IgG N-glycomic changes with statistical significance (p
< 0.05) for the pooled samples from three Chinese Muslim ethnic minorities with HDC; and the lower level of GP20 (FA2FG2S1) were observed to be unique to the Han Chinese with HDC. In the pooled samples from the three Chinese Muslim ethnic minorities, HDC patients presented increased core-fucosylation with bisecting GlcNAc of IgG (GP6 and DG54), but decreased sialylation (GP16, GP18, and DG3) and monogalactosylated (GP16) and digalactosylated (GP18) core-fucosylated glycans without bisecting GlcNAc of IgG, when compared with HTN patients. In comparison with T2DM patients, HDC patients showed decreased galactosylation, sialylation, and core-fucosylation without bisecting GlcNAc of IgG (GP16 and GP18), but increased mannose structures of IgG (GP5). Additionally, compared to healthy individuals, HDC patients presented increased core-fucosylation with bisecting GlcNAc (GP6) and mannose structures of IgG (GP5). In the Han Chinese, the lower level of core-fucosylated galactosylated glycan containing sialic acid of IgG (GP20) was observed in the HDC compared with HTN/T2DM/healthy individuals, while sialylated glycans (reduced relative abundance of GP20 and increased relative abundance of GP24 [FA2BG2S2], DG2 [FBGS/(FBG + FBGS)], and DG53 [FG2n
)]) presented inconsistent trends in HDC patients. Specifically, the common characteristic of these HDC-associated sialylated glycans is the presence of bisecting N-GlcNAc, and the increase of this structural feature in HDC outweighed the effects of a decrease in sialylation, which is in agreement with the alterations of sialyation observed in the study on systemic lupus erythematosus [29
Fucosylation plays key roles in the function of IgG and highly fucosylated IgG shows a reduced magnitude of antibody-dependent cellular cytotoxicity (ADCC), which is a result of IgG-subclass IgG1 binding to each of the different Fc receptors [41
]. Bisecting GlcNAc is the central branch of N-glycans, which is biosynthesized by the glycosyltransferase GnT-III encoded by the MGAT3 gene [42
]. The presence of bisecting GlcNAc in N-glycans could suppress various terminal modifications (fucosylation, sialylation, and others) of N-glycans [42
], affecting the structure of glycans exerting noteworthy effects on inflammatory responses or immune function. IgG carrying glycans with bisecting GlcNAc was reported to have pronouncedly increased affinity to FcγRIIIa leading to an enhancement in ADCC, whereas fucosylation without bisecting GlcNAc of IgG can reduce ADCC [37
]. Previous studies showed that such increased fucosylation with bisecting GlcNAc was associated with T2DM [15
]. We also observed similarly increased fucosylation in structures with bisecting GlcNAc (GP6), and decreased fucosylation in structures without bisecting GlcNAc (GP16, GP18, and GP20) in the HDC patients, compared with HTN and T2DM patients. The increased fucosylation in structures with bisecting GlcNAc of IgG N-glycans could lead to the pro-inflammatory status of the body [37
], thereby contributing to the progression of HTN or T2DM towards HDC. Thus, the present results indicate that the individuals with higher levels of fucosylated structures with bisecting GlcNAc of IgG may increase the risk for HDC, and that pathophysiological states of individuals with HDC are conditioned on a chronic inflammatory process.
The level of galactosylation has an essential impact on the inflammatory activities of IgG; lack of galactosylation was shown to associate with the pro-inflammatory autoimmune responses through activation of the complement cascade [43
]. The down-regulation of galactosyltransferase activity in plasma cells and the activation of complement cascades were identified as potential mechanisms for the changes in IgG galactosylation [43
]. As a modulator of inflammatory activity, high galactosylation of IgG Fc N-glycan promotes the association of the inhibitory receptor FcγRIIb and the lectin-like receptor dectin-1, which blocks the pro-inflammatory effector functions of C5aR and CXCR2, and enhances the anti-inflammatory activity of antigen-specific IgG1 immune complexes [44
]. Previous studies have suggested that the decrease in galactosylation of IgG N-glycans play potential roles in the development and progression of chronic pro-inflammatory diseases, including HTN, T2DM, and dementia [14
], which are in agreement with the alterations of IgG N-glycosylation patterns observed in the HDC patients in the present study. The decreased IgG galactosylation found in HDC presented a similar pattern found in the previous studies and thus our finding further supports the hypothesis that a decrease in galactosylation is a general feature of multiple chronic diseases associated with decreased anti-inflammatory/immunosuppressive potential of circulating IgG.
Besides fucosylation and galactosylation, sialylation mediates partly the inflammatory function of IgG. IgG shows different pro- and anti-inflammatory potentials on the basis of different Fcγ receptors that IgG preferentially binds to, and the affinity of the receptor depends on the composition of the glycans attached to the 297 asparagine [46
]. The addition of sialic acid to IgG can decrease its affinity for FcγRIII, thereby reducing the degree of ADCC, converting its function from pro- to anti-inflammatory [47
]. In the two comparison groups (HDC versus HTN, HDC versus T2DM), we observed that patients with HDC showed a significant decrease in sialylation (GP16, GP18, and GP20), indicating that sialylation is correlated with HDC through decreasing adaptive pro-inflammatory actions of IgG. However, sialylation with bisecting GlcNAc showed the reduced relative abundance in HDC patients, which was consistent with the suppression of core fucosylation expression due to the presence of bisecting GlcNAc. Bisecting GlcNAc inhibits the biosynthesis and modification of sialic acid of terminal epitopes in N-glycans, which results in the relative abundance of sialylation (GP16, GP18, GP20, GP24, DG2, DG3, and DG53) presenting inconsistent trends in HDC patients. Additionally, we found the decrease of GPs containing galactosylated sialylated glycan structures (GP16, GP18, and GP20) in HDC patients. Since terminal sialic acid residues are covalently attached to galactosylated glycans, the decrease of galactosylated sialylation could be a consequence of a decrease in galactosylation [48
], which could provide a plausible explanation for the phenomenon of the co-directional changes of sialylation and galactosylation shown in the HDC patients. It is worthwhile to investigate the potential of the level of sialylation and galactosylation of IgG for monitoring the progression from HTN or T2DM towards HDC. Thus, future prospective cohort studies are called for to explore if there is a stepwise decrease trend in levels of sialylation and galactosylation of IgG during progression from healthy individuals to HTN or T2DM and then to HDC.
Among the different Chinese ethnic minorities, the similarities and differences in the glycosylation patterns were found in both the HTN and T2DM patients, compared with the HDC patients, suggesting that those individuals who have developed T2DM or HTN do not show consistent alterations of IgG N-glycans. More work is required on understanding alterations of IgG N-glycans in HTN or T2DM individuals to see if their alterations over time show an inclination towards HDC. Such IgG N-glycans profiling will help to contribute towards developing potential glycan biosignatures for monitoring the progression from HTN or T2DM towards HDC.
The AUCs of the combined models, based on the glycan traits and the baseline characteristics, demonstrate higher discriminative performance to help discern between HDC, HTN, T2DM, and healthy individuals than the models based on either the glycan traits or the baseline characteristics alone. IgG N-glycan profiles reflect the integrative effects of genetic, metabolic, and environmental factors, contributing significantly towards their discriminative potential [11
]. Although the addition of glycan traits to the baseline models did not substantially improve the discrimination in all the combined models, the glycan based models constructed based on IgG N-glycans alone displayed enough discrimination power and all the combined models showed improved discrimination performance. These results suggest that IgG N-glycan profiles may contribute significantly to much of the combined risk of these characteristics, where dynamic and population-specific IgG N-glycomic changes can serve as functional effectors of the disease potentially increasing the diagnostic accuracy for complex diseases. The IgG N-glycosylation profiles associated with HDC described in this study may thus help to shed light on future studies investigating their biomarker potential for personalized monitoring and prevention of the progression from HTN or T2DM towards HDC.
The strength of this study is that we evaluated IgG N-glycomes in relation to HDC and explored similarities and differences in the IgG N-glycosylation profiles for the first time, particularly in different Chinese ethnic groups, including the Chinese Muslim minorities. The alterations of IgG N-glycosylation in HDC patients found in this study suggest a theoretical foundation and present practical direction for future studies to further elaborate the association of IgG glycosylation with HDC. These suggestions are, however, built upon the constraints of this study. Firstly, due to the small sample size of HDC in each ethnic minority in the present study, it is unfeasible to analyze the association of IgG N-glycans with HDC in each of the Muslim minorities separately. Consequently, the same health condition groups from the three Chinese Muslim ethnic minorities were pooled together for the analysis. Furthermore, the Han subjects included in the study were not co-resident in the Xinjiang Autonomous Region since the Muslim minorities were recruited from the isolated rural areas in Xinjiang, where the respective Muslim minority “exclusively” inhabit for generations without co-resident Han or other ethnic groups. Additionally, it is difficult to match the Han subjects with the Muslim minorities in terms of their profile of demographic variables due to the relatively small sample size of the respective health condition group of the Muslim minorities. To compensate for the potential impact of unmatched demographic variables on the results, we adjusted the potential confounding factors in the logistic regression analyses. Thirdly, the case-control nature of the present study was not able to provide the information about a causal relationship between the altered IgG glycosylation and the progression of HDC. A previous study reported that the polymorphisms at six loci (i.e., FUT8
, and SLC9A9
) affect plasma levels of N-glycans [49
], which could be used as the instrumental variables for future Mendelian randomization (MR) studies investigating the causal relationship between the altered IgG glycosylation and HDC. Fourthly, the information on medical treatment of the patients with HTN and/or T2DM was not completely collected. Consequently, the potential impact of medical treatment for HTN and/or T2DM on the associations between HDC and IgG glycans was unable to be addressed in this current study. Lastly, only the five-fold cross-validation of the model is performed, external validation of model performance is still needed in further studies.
In the current study, the IgG N-glycans were analyzed by HILIC-UPLC method, which is a profiling method for total IgG N-glycosylation. The detailed differences between the glycosylation profiles of the individual IgG subclasses associated with HDC remain to be investigated, which would be performed in our coming study. Future prospective studies with larger samples in multiracial populations and/or MR studies examining causal inference are warranted to validate the potential of IgG N-glycosylation biomarkers for personalized monitoring and prevention of HDC. Clinical exploitation of IgG N-glycan biomarkers will be facilitated by the existing broad application in clinical laboratories of UPLC. This study shows the potential clinical application of N-glycans as dynamic biomarkers (e.g., of routine diagnostic and prophylactic test for HDC patients). Future longitudinal and prospective studies would provide a better understanding of the transition from HTN or T2DM to HDC and allow us to generate glycan-based markers for use in earlier detection of the risk of progression towards HDC. As such, healthy lifestyle intervention and therapeutic decision-making could be delivered timely for the HDC prophylaxis from the perspectives of predictive, preventive and personalized/precision medicine.