Evolution of diagnostic criteria and new insights into clinical testing in mixed connective tissue disease; anti-survival motor neuron complex antibody as a novel marker of severity of the disease

Abstract

Mixed connective tissue disease (MCTD) is an autoimmune disorder characterized by a combination of clinical features from systemic lupus erythematosus, systemic sclerosis, and inflammatory muscle disease, along with the presence of positive anti-U1-ribonucleoprotein (U1-RNP) antibodies. The exact etiology of the disease remains unclear, but it is believed to involve vascular damage within the context of heightened autoimmune responses. Consequently, Raynaud’s phenomenon and pulmonary arterial hypertension are observed in patients with MCTD. While specific biomarkers for MCTD have not yet been identified, the recent study of the utility of anti-survival motor neuron complex (SMN) antibodies in MCTD suggests a promising avenue for further research and the accumulation of additional evidence.

Keywords:

• Mixed connective tissue disease
• anti-survival motor neuron complex antibodies
• survival motor neuron complex
• biomarker

1. Introduction

Mixed Connective Tissue Disease (MCTD) is an autoimmune disease characterized by elevated anti-U1-ribonucleoprotein (U1-RNP) antibody levels and a combination of features from systemic lupus erythematosus, systemic sclerosis, and inflammatory muscle disease, often presenting with minimal clinical manifestations. Raynaud’s phenomenon is universally observed in all patients, and vascular-related organ damage, such as pulmonary arterial hypertension, is frequently encountered. Despite the prevalence of MCTD, limited research has been conducted, and as of now, there is no specific treatment tailored to the disease. Current management relies on the use of drugs employed in systemic lupus erythematosus and systemic sclerosis, with a scarcity of clinical trials dedicated to MCTD.

Autoantibodies are identified in various autoimmune diseases. In most instances, the production of autoantibodies is considered to be a concomitant phenomenon of autoimmune disease pathology, and its etiological significance remains unclear. Nevertheless, autoantibodies play a crucial role in the diagnosis and categorization of autoimmune diseases, serving as essential biomarkers widely utilized in daily clinical practice. For example, antibodies such as anti-dsDNA antibodies in systemic lupus erythematosus and anti-CCP antibodies in rheumatoid arthritis hold significant diagnostic value. In addition, certain antibodies, for instance, the anti-MDA-5 antibody, strongly indicate the prognosis of the disease.

The anti-U1-RNP antibody is essential for diagnosing MCTD. While the positive predictive value for diagnosing MCTD may not be consistently high, the sensitivity is 100%, and the diagnosis of MCTD is contingent upon the presence of this antibody. Unlike other autoimmune diseases, the diagnosis of MCTD relies on the presence or absence of specific antibodies, making it a rare condition where the disease concept is formulated based on an antinuclear antibody profile.

2. Evolution of MCTD classification and diagnostic criteria

The concept of MCTD was initially introduced in 1972 through a case series involving 25 patients exhibiting features of systemic lupus erythematosus, systemic sclerosis, and inflammatory muscle disease, along with elevated titers of anti-U1-RNP antibodies [1]. However, during that period, MCTD was not clearly distinguished from undifferentiated connective tissue disease (UCTD), which lacked typical symptoms and could not be categorized as a specific autoimmune disease. Recognition of MCTD in the West remained limited. Over time, it has been consistently demonstrated that MCTD is characterized by clinical features such as Raynaud’s phenomenon and puffy fingers, coupled with distinctive findings such as a heightened incidence of pulmonary arterial hypertension and trigeminal lesions. Consequently, MCTD is now acknowledged as an independent disease concept separate from UCTD [2–4].

For a considerable duration, internationally standardized criteria for the diagnosis and classification of Mixed Connective Tissue Disease (MCTD) were not established. Instead, criteria proposed by Sharp, Alarcon-Segovia, Kahn, and Kasukawa were utilized [5,6]. Among these, Sharp’s classification criteria, initially published, comprised major and minor criteria, and a combination of these was suggested for diagnosing MCTD (Table 1). In the meantime, the criteria presented by Alarcon-Segovia and Kahn were more streamlined, relying solely on multiple clinical findings in conjunction with a positive anti-U1-RNP antibody. All these diagnostic criteria are grounded in three or four clinical signs, alongside serological tests. Moreover, the Japanese MCTD study group (Kasukawa et al.) devised diagnostic criteria founded on clinical information encompassing features of systemic lupus erythematosus, systemic sclerosis, and inflammatory muscle disease [7]. These criteria have largely served as the foundation for the present diagnostic criteria, undergoing revisions in 2004 and 2019, which introduced additional criteria. The current criteria were expanded to incorporate ‘pulmonary arterial hypertension’, ‘aseptic meningitis’, and ‘trigeminal neuropathy’, which are organ disorders distinctive to MCTD [8]. More importantly, the criteria are based on items from the Sharp criteria, 1996 criteria, and 2004 criteria, developed through expert consensus, and evaluated using clinical data from typical and borderline cases of MCTD. A final draft has been established after receiving public comments from the Japan College of Rheumatology and other organizations. Additionally, these criteria have been independently validated by another cohort and compared to existing criteria, thus refining the concept of MCTD. As shown in Table 1, diagnosis under the criteria is established when one common symptom, one immunologic symptom, and at least one characteristic organ lesion are concurrently present. Alternatively, diagnosis is made when one common symptom, one immunologic symptom, and two or more overlapping symptoms from items A, B, and C are evident (Table 1). The novel criteria exhibit a sensitivity of 90.6% and a specificity of 98.4%, surpassing the accuracy of the previous criteria. The international acknowledgment of the diagnostic criteria is deemed crucial for future advancements.

Table 1. Comparison of diagnostic criteria for MCTD.

Tanaka et al.*	Sharp et al.**	Alarcón-Segovia et al.***	Kahn et al.****
I. Common manifestations	Major criteria	Serological criteria	Serological criteria
1. Raynaud’s phenomenon	1. Myositis	Anti-RNP Ab positive	High titer anti-RNP Ab
2. Puffy fingers and/or swollen hands	2. Pulmonary involvement:
II. Immunological manifestation	a. Diffusion of normal values
Positivity for anti-U1 RNP Ab	b. Pulmonary hypertension	Clinical criteria	Raynaud’s phenomenon
III. Characteristic organ involvement	c. Proliferative vascular lesions on lung biopsy	1. Edema in hands
1. Pulmonary arterial hypertension	3. Raynaud’s phenomenon or esophageal hypomotility	2. Synovitis	Clinical criteria
2. Aseptic meningitis	4. Swollen hands	3. Myositis	1. Synovitis
3. Trigeminal neuropathy	5. Anti-U1 RNP Ab positive and anti-Sm negative	4. Raynaud’s phenomenon	2. Myositis
IV. Overlapping manifestations	and Anti-ENA positive	5. Acrosclerosis	3. Swollen fingers
A. Systemic lupus erythematosus-like manifestations
1. Polyarthritis	Minor criteria
2. Lymphadenopathy	1. Alopecia
3. Malar rash	2. Leukopenia
4. Pericarditis or pleuritis	3. Anemia
5. Leukopenia or thrombocytopenia	4. Pleuritis
B. Systemic sclerosis-like manifestations	5. Pericarditis
1. Sclerodactyly	6. Arthritis
2. Interstitial lung disease	7. Trigeminal neuropathy
3. Esophageal dysmotility or dilatation	8. Malar rash
C. Polymyositis/Dermatomyositis-like manifestations	9. Thrombocytopenia
1. Muscle weakness	10. Mild myositis
2. Elevated levels of myogenic enzymes	11. History of swollen hands
3. Myogenic abnormalities on electromyogram

* At least one common manifestation, immunological manifestation, and at least one characteristic organ involvement; or when a patient meets all the following: at least one common manifestation, immunological manifestation, and at least one feature each in 2 or more from items A, B, and C in overlapping manifestations.

** At least 4 major criteria plus the presence of anti-U1-RNP antibody; or 2 major criteria among criteria 1 to 3 plus 2 minor criteria along with anti-U1-RNP antibody.

*** At least 3 clinical criteria, including either synovitis or myositis, along with the presence of anti-U1-RNP antibody.

**** At least two clinical criteria, in addition to Raynaud’s phenomenon, along with the presence of anti-U1-RNP antibody.

The cornerstone in both the classification and diagnostic criteria is the anti-U1-RNP antibody, which is an obligatory component in all MCTD classification and diagnostic criteria utilized thus far (Table 1). Unlike other criteria, none necessitate such specific autoantibodies, underscoring that MCTD is a disease concept firmly grounded in the presence of anti-U1-RNP antibodies.

3. Microvascular abnormality in patients with MCTD

Despite the unknown underlying mechanism of MCTD, the existence of Raynaud’s phenomenon and pulmonary arterial hypertension, indicative of typical clinical signs, implies that vasculopathy plays a pivotal role in MCTD pathogenesis. Nailfold capillary video capillaroscopy (NVC) proves beneficial in assessing microvascular lesions related to this condition [9,10]. NVC enables real-time and minimally invasive evaluation of vascular lesions in individuals with autoimmune diseases and has been extensively studied, particularly in cases of systemic sclerosis (Figure 1). The application of NVC has been instrumental in appraising vascular lesions in patients with autoimmune diseases [11,12]. Nonetheless, there is limited research and a scarcity of reports on NVC abnormalities in MCTD. The frequency of these abnormalities varies due to studies with a small number of patients and analyses that include a combination of cases with both long disease duration and post-treatment situations [13–16]. Consequently, we conducted an assessment of vascular damage through NVC in untreated MCTD to elucidate its significance [17].

Figure 1. Typical Nailfold Videocapillaroscopy Finding in Patient with MCTD. Blue and black arrows are used to denote giant capillaries and microhemorrhages, respectively.

While NVC abnormalities are commonly associated with the Raynaud phenomenon, it is surprising to note that the frequency of NVC abnormalities in MCTD is only 41.3%, a markedly lower figure compared to systemic sclerosis, where the frequency is 88.6% [17]. Moreover, it is established that immunosuppressive therapy does not lead to improvements in NVC abnormalities in systemic sclerosis. However, in the case of MCTD, there was an observed improvement in NVC abnormalities after one year of immunosuppressive therapy. This suggests a distinct difference in the significance of microangiopathy between MCTD and systemic sclerosis. This divergence could stem from the fact that in systemic sclerosis, Raynaud’s phenomenon tends to manifest after irreversible morphological changes in the nailfold capillaries have occurred. In contrast, in MCTD, circulatory insufficiency may arise before the onset of irreversible morphological changes. This distinction may be attributed to various factors, including immunological mechanisms and heightened reactivity of the vessel wall [12, 18,19]. On the other hand, when juxtaposed with systemic lupus erythematosus, MCTD tends to be more frequently associated with the Raynaud phenomenon and pulmonary arterial hypertension as clinical manifestations. In alignment with these clinical differences, the prevalence of NVC abnormalities were also found to be less prevalent in systemic lupus erythematosus in comparison to MCTD. Nevertheless, upon examining the response of NVC abnormalities to treatment, it was observed that vasculopathy disappeared in all patients with systemic lupus erythematosus after treatment, mirroring the outcomes in MCTD. This suggests that, despite differences in the frequency of microvascular damage between MCTD and SLE, the underlying mechanism for the development of vascular damage may be equivalent.

Moreover, an analysis comparing NVC abnormalities with organ damage in MCTD revealed an association with the presence of pulmonary arterial hypertension, implying that pulmonary arterial hypertension in MCTD might be rooted in reversible microvascular damage. Remarkably, there were instances where patients initially diagnosed without pulmonary arterial hypertension, subsequently monitored without treatment, later developed abnormal NVC and pulmonary arterial hypertension simultaneously. This suggests that NVC in MCTD could serve as a predictive factor for pulmonary arterial hypertension [17].

4. Anti- survival of motor neuron (SMN) complex antibody

The sensitivity of the anti-U1-RNP antibody is 100%, making it an unparalleled marker for sensitivity. However, its specificity is not as high, prompting a need for the identification of an antibody with greater specificity for MCTD. Previously, three patients exhibiting myositis and positive for autoantibodies against the Survival of Motor Neuron (SMN) and its complex (Gemin 2-7), referred to as anti-SMN complex antibodies, were reported among 1996 patients with autoimmune diseases [20]. Subsequent studies revealed that anti-SMN complex antibodies tend to coexist with anti-U1-RNP antibodies and are seldom positive in cases negative for anti-U1-RNP [21,22]. Building upon these findings, we conducted a comprehensive analysis of autoantibodies through immunoprecipitation in MCTD to explore the clinical significance of anti-SMN complex antibodies in this context [23].

In the conducted study, approximately 40% of MCTD patients tested positive for anti-SMN complex antibodies. In contrast, only about 10% of patients diagnosed with systemic lupus erythematosus or systemic sclerosis, and who were positive for anti-U1-RNP antibodies, exhibited positivity for anti-SMN complex antibodies (Table 2). In these cases, anti-SMN complex antibodies demonstrated a notable specificity of 91.2% for diagnosing MCTD (with a sensitivity of 35.8%). The presence of this antibody suggested the likelihood of MCTD at a considerably high rate. This suggests that anti-SMN complex antibodies may serve to compensate for the relatively lower specificity of anti-U1RNP antibodies [23].

Table 2. The prevalence of anti- SMN complex antibody, anti- Sm antibody, and anti- SS-a antibody among patients with MCTD, SLE, and SSc.

Antibodies	MCTD (n = 67)	SLE (n = 74	SSc (n = 17)
Anti-SMN complex Antibody, (%)	35.8	8.1	11.8
Anti-Sm Antibody, (%)	19.4	60.8	17.6
Anti-SS-A/Ro60 Antibody, (%)	46.3	68.9	23.5

MCTD: mixed connective tissue disease; SLE: systemic lupus erythematosus; SSc: systemic sclerosis; SMN: survival motor neuron.

Significantly, the comparison between MCTD patients positive for anti-SMN complex antibodies and those negative for these antibodies unveiled noteworthy differences in their clinical presentations. Particularly, MCTD patients positive for anti-SMN complex antibodies exhibited a more unfavorable prognosis compared to their negative counterparts. In more than 95% of the positive cases, complications involving either pulmonary arterial hypertension or interstitial lung disease were identified, deemed as contributing factors to the poor prognosis. Moreover, concerning the microvasculopathy discussed earlier in this review, it was observed that 70% of the positive cases exhibited abnormalities in NVC. This suggests that anti-SMN complex antibodies are valuable not only for diagnostic purposes but also for assessing severity of MCTD. While previous reports have indicated an association between high-titer positivity for anti-Ro52 and anti-U1-RNP antibodies with interstitial lung disease in MCTD, our study revealed that anti-SMN complex antibodies played a more substantial role in determining severity compared to high-titer positivity for anti-Ro52 or anti-U1-RNP antibodies [24,25].

Conversely, the pathogenicity of the SMN complex (SMN and Gemin 2-7) itself remains uncertain. SMN proteins form complexes and play crucial roles in several fundamental cellular homeostatic pathways such as spliceosome assembly, and ribonucleoprotein biosynthesis. (Figure 2). It plays a crucial role in the organization of snRNPs, including U1-RNP and Sm, and homozygous mutations in SMN1 gene are known to cause spinal muscular atrophy [26]. Similar to anti-U1-RNP antibodies, there might be no direct relationship between the function of the antigen itself and the production of antibodies in the case of anti-SMN complex antibodies. Anti-SMN complex antibodies in MCTD were reported at approximately the same time by another research group, indicating positive results in around 60% of MCTD patients and a high incidence of complications, including myocarditis [27]. Further studies are warranted to explore the sensitivity and specificity of anti-SMN complex antibodies for diagnosis, their association with organ damage, and variations in antibody titers before and after treatment. These aspects are crucial for a comprehensive understanding of the clinical implications and potential applications of these antibodies in the context of MCTD. We are currently developing assay systems, including ELISA, which are undergoing validation. This process aims to prepare these systems for widespread use in the future.

Figure 2. Schematic representation of the survival motor neuron complex.

5. Conclusion

We have provided an overview of the pathogenesis of MCTD, emphasizing autoantibodies, including insights from our own research. In Europe and the United States, MCTD is not yet widely recognized, and there have been limited clinical studies on MCTD compared to other autoimmune diseases. Nonetheless, specific antibodies, as detailed in this paper, have been identified. It is anticipated that further establishment and dissemination of the disease concept of MCTD will contribute to unraveling the molecular basis of MCTD and the development of disease-specific therapies.

Author contributions

Satoshi Kubo contributed to the writing of the manuscript. Yoshiya Tanaka contributed to the overall review. All authors read and approved the final manuscript.

Disclosure statement

Satoshi Kubo has received speaking fees from Eli Lilly, GlaxoSmithKline, Bristol-Myers, Abbvie, Eisai, Pfizer, Astra-Zeneca and also research grants from Daiichi-Sankyo, Abbvie, Boehringer Ingelheim, and Astellas. Yoshiya Tanaka has received consulting fees, speaking fees, and/or honoraria from Abbvie, Daiichi-Sankyo, Chugai, Takeda, Mitsubishi-Tanabe, Bristol-Myers, Astellas, Eisai, Janssen, Pfizer, Asahi-Kasei, Eli Lilly, GlaxoSmithKline, UCB, Teijin, MSD, and Santen, and also research grants from Mitsubishi-Tanabe, Takeda, Chugai, Astellas, Eisai, Taisho-Toyama, Kyowa-Kirin, Abbvie, and Bristol-Myers.

Additional information

Funding

This work was supported by JSPS KAKENHI, and the University of Occupational and Environmental Health, Japan, through UOEH Grant for Advanced Research.