Best Practice & Research Clinical Rheumatology 30 (2016) 95e111

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Best Practice & Research Clinical

Rheumatology
journal homepage: www.elsevierhealth.com/berh

Mixed connective tissue disease

Ragnar Gunnarsson a, *, Siri Opsahl Hetlevik b, Vibke Lilleby a,
Øyvind Molberg a, b
a
Rheumatology Unit, Oslo University Hospital Rikshospitalet, Oslo, Norway
b
Institute of Clinical Medicine, University of Oslo, Oslo, Norway

a b s t r a c t
Keywords:
Mixed connective tissue disease The concept of mixed connective tissue disease (MCTD) as a
Systemic sclerosis separate connective tissue disease (CTD) has persisted for more
Myositis than four decades. High titers of antibodies targeting the U1 small
Polymyositis nuclear ribonucleoprotein particle (U1 snRNP) in peripheral blood
Dermatomyositis are a sine qua non for the diagnosis of MCTD, in addition to distinct
Systemic lupus erythematosus
clinical features including Raynaud's phenomenon (RP), “puffy
Pulmonary hypertension
Interstitial pulmonary fibrosis
hands,” arthritis, myositis, pleuritis, pericarditis, interstitial lung
Genetics disease (ILD), and pulmonary hypertension (PH). Recently,
U1 small nuclear ribonucleoprotein population-based epidemiology data from Norway estimated the
point prevalence of adult-onset MCTD to be 3.8 per 100,000 and
the mean annual incidence to be 2.1 per million per year, sup-
porting the notion that MCTD is the least common CTD. Little is
known about the etiology of MCTD, but recent genetic studies have
confirmed that MCTD is a strongly HLA (human leukocyte anti-
gen)-linked disease, as the HLA profiles of MCTD differ distinctly
from the corresponding profiles of ethnically matched healthy
controls and other CTDs.
In the first section of this review, we provide an update on the
clinical, immunological, and genetic features of MCTD and discuss
the relationship between MCTD and the other CTDs. Then we
proceed to discuss the recent advances in therapy and our current
understanding of prognosis and prognostic factors, especially
those that are associated with the more serious pulmonary and
cardiovascular complications of the disease. In the final section, we
discuss some of the key, unresolved questions related to anti-RNP-

* Corresponding author. Rheumatology Unit, Oslo University Hospital, P.O. Box 4950 Nydalen, 0424 Oslo, Norway. Tel.: þ47
911 76 839; fax: þ47 67 209324.
E-mail address: ragunnar@gmail.com (R. Gunnarsson).

http://dx.doi.org/10.1016/j.berh.2016.03.002
1521-6942/© 2016 Published by Elsevier Ltd.
96 R. Gunnarsson et al. / Best Practice & Research Clinical Rheumatology 30 (2016) 95e111

associated diseases and indicate how these questions may be

approached in future studies.
© 2016 Published by Elsevier Ltd.

Introduction

The concept of mixed connective tissue disease (MCTD) as a separate immune-mediated connective
tissue disease (CTD) was first introduced by Gordon C. Sharp and coworkers > 40 years ago, but there is
still no consensus regarding the disease definitions, the classification criteria, or the relationship with
other CTDs [1]. Some rheumatologists argue that MCTD is a distinct disease entity; others believe that it
represents an overlap syndrome or an early and unspecific phase of an evolving, more distinct CTD;
however, few studies still disregard the whole concept [2e4].
In their initial report, Sharp and coworkers described MCTD as a mild disease with favorable out-
comes and an excellent response to oral prednisolone [1]. This description holds true for some patients,
but the overall impression from published cohorts and case series is that MCTD is a complex disease,
with large interindividual variations in clinical features, responses to therapy, and outcomes [5e12].
The disease appears in all age groups, but the peak incidence of MCTD is around 40 years [13]. Previous
studies have reported that 7e23% of the total MCTD population have a juvenile onset [10,14]. Whether
there are any systematic differences between adult and juvenile MCTD (JMCTD) remains unclear.
To date, there are no uniform guidelines for evaluating patients presenting with systemic disease
features and a high titer of serum autoantibodies directed against the U1 ribonucleoprotein (anti-RNP),
and there is no international consensus on how, when, and in whom MCTD should be diagnosed
[8,9,15]. Nevertheless, most physicians working in the CTD clinics would probably agree that the
diagnosis of MCTD should be considered in an anti-RNP-positive patient presenting with Raynaud's
phenomenon [1,10,16,17], diffuse hand edema (“puffy hands”), and at least two of the following fea-
tures: arthritis, myositis, leukopenia, esophageal dysmotility, pleuritis, pericarditis, interstitial lung
disease (ILD) [18e24], or pulmonary hypertension (PH) [5,7e9,25,26]. However, none of these features
are unique to MCTD. They can also be observed in patients with systemic lupus erythematosus (SLE),
systemic sclerosis (SSc), and polymyositis/dermatomyositis (PM/DM), the three CTDs that mostly
resemble MCTD. Diagnosing MCTD in clinical practice is therefore an issue of pattern recognition and
clinical decision with rich possibilities for different opinions and practices across the rheumatology
communities.
The four coexisting criteria sets for MCTD further complicate this issue (Supplementary Table 1).
Even though all these four criteria sets were originally presented as diagnostic criteria, they are
distinctly different (Table 1), and within a clinical MCTD population, they do not capture the same
patients (Fig. 1). Three of the proposed criteria sets [27e29] were first presented at an international
conference on MCTD held in Japan in 1986 and the last criteria set was published in France in 1991 [30].
Only two of these four criteria sets have been regularly used for research purposes in adult populations:
the Alarco n-Segovia criteria and the Kasukawa criteria [28,29]. The Kasukawa criteria have mostly been
used in juvenile MCTD (JMTCD) [12,28,29]. Sharp's modified criteria set has not been widely used for
several reasons, the most obvious being their complexity and the need for autoantibody data that are
no longer available from routine immunology laboratories (see Table 1). The limited use of the Kahn
criteria may be related to accessibility as the criteria were originally presented in French, and the fact
that they closely resembled one of the already published criteria sets (Table 1).
Compared to the other CTDs, the research activity on MCTD has been relatively low from the late
1990s. Unfortunately, as a result, a large proportion of the standard textbook references on MCTD
include >20 years old and originate from relatively small and potentially skewed single-center hospital
cohorts [5e12]. An extensive study of MCTD has not been conducted recently due to the following four
interrelated reasons: (1) the controversies regarding the concept may have dampened the enthusiasm
of some researchers; (2) the apparent rarity of the disease, with a concomitant lack of population-
based epidemiology estimates, has made it difficult to plan and design MCTD studies; (3) the lack of
 R. Gunnarsson et al. / Best Practice & Research Clinical Rheumatology 30 (2016) 95e111 97

Table 1
Overview of the items included in the four published MCTD criteria sets.

Sharp Kasukawa et al. Alarcon-Segovia and Kahn and

Villareal Appelboom

Immunological marker anti-U1RNP anti-nRNP anti-RNP anti-RNP

(obligatory) -high titer anti-ENA -high titer (by HA -high titer by
Positive anti-Sm assay) immune-diffusion
excludes definite MCTD or
immunoblot (U1
68kd)
Raynaud's -one of four major -one of two obligatory -one of five clinical -obligatory criteria
phenomenon criteria criteria criteria
Swollen/‘puffy’ hands -one of four major -one of two obligatory -one of five clinical -one of three clinical
criteria criteria criteria criteria
Joint involvement Arthritis Polyarthritis Synovitis Synovitis
-one of 11 minor criteria -one of five SLE-like -one of five clinical -one of three clinical
findings criteria criteria
Muscle involvement Myositis (mild) 1 of muscle weakness, Myositis (lab. or biopsy Myositis
-one of 11 minor criteria elevated CK, myogenic proven) -one of three clinical
EMG -one of five clinical criteria
-disease category (2/3 criteria
needed)
Peripheral sclerosis Sclerodactyly Sclerodactyly Acrosclerosis Not included
-can substitute swollen -one of three SSc-like (þ/scleroderma)
hands as one of four findings -one of five clinical
major criteria criteria
Lung involvement DLCO < 70%, PAH or Pulmonary fibrosis, Not included Not included
lung biopsy with vital capacity < 80% or
proliferative vascular DLCO < 70%
lesions -one of three SSc-like
-one of four major findings
criteria
Esophageal disease Hypomotility Hypomotility or Not included Not included
-can substitute dilatation
Raynaud's as one of four -one of three SSc-like
major criteria findings
Serositis Pleuritis, Pericarditis Pericarditis or pleuritis Not included Not included
-two of 11 minor -one of five SLE-like
criteria findings
Hematopathology Leukopenia, Anemia, Leukocytopenia or Not included Not included
Thrombocytopenia thrombocytopenia
-three of 11 minor -one of five SLE-like
criteria findings
Other Alopecia, Trigeminus Lymphadenopathy, Not included Not included
neuropathy, Malar rash, Facial erythema
History of swollen -two of five SLE-like
hands criteria
-four of 11 minor criteria

clear disease definitions has made it difficult to delineate MCTD from the other CTDs; and (4) the lack of
internationally accepted consensus criteria for disease classification has reduced the possibility of
comparative studies.
Recently, we focused on the concept of MCTD and decided to tackle some of the unresolved issues
related to the disease. Initially, we took advantage of the research possibilities provided by the orga-
nizational structure of the Norwegian Public Health System. In this system, care for CTDs is a specialist
task. All CTD cases are therefore referred to, and routinely followed by, rheumatologists based at public
hospitals. This organizational structure makes it possible to set up national level CTD case-finding
strategies based on ICD-10 (10th revision of The International Statistical Classification of Diseases
and Related Health Problems) searches across all hospital administrative databases and manual review
of electronic patient journals (EPJs). Using these strategies, we were able to provide the first
98 R. Gunnarsson et al. / Best Practice & Research Clinical Rheumatology 30 (2016) 95e111

A. Adult MCTD

Kasukawa Sharp Alarcon-Segovia

8 13
75

2 1
1

B. Juvenile MCTD

Kasukawa Alarcon-Segovia
Alarcon-Segovia

12 78 10

Fig. 1. AeB: Venn diagram: overview of the MCTD criteria in cohorts of adult and juvenile MCTD patients.

population-based data on the epidemiology of MCTD [13]. The study cohort included every adult
patient (18 years) in Norway who was (A) diagnosed with MCTD by a rheumatologist, (B) who met the
 n-Segovia and Villareal or those of Kasukawa, and for
modified Sharp's criteria or the criteria of Alarco
whom (C) other CTD diagnoses were excluded by consensus by the study physicians. We found that the
point prevalence of MCTD in Norway by 2008 was 3.8 per 100,000 adults and estimated the mean
annual incidence of an adult-onset MCTD to be 2.1 per million per year. The female-to-male ratio was
3.3. The observed female-to-male ratio in our cohort was lower than in most other studies, including a
recent large (n ¼ 280) Hungarian cohort study that reported a ratio of 12.3. There are currently no
population-based studies on juvenile MCTD, but the available data indicate that the female-to-male
ratio in this age group may be in the range of 3e6 [12,31,32]. In the Norwegian cohort study, the
prevalence of MCTD was similar across the three criteria sets of MCTD [27e29]. The vast majority of the
patients (97.3%) met at least two of the three MCTD criteria and 75% met the three criteria sets applied
(Fig. 1A). There were, however, differences, particularly in relation to patients with ILD [33].

Update on the immunological and clinical features of MCTD

Immunological features

High titers of antibodies targeting the U1 small nuclear ribonucleoprotein particle (U1 snRNP) in
peripheral blood are a sine qua non for the diagnosis of MCTD (Table 1). These antibodies were orig-
inally detected by immunodiffusion. They were first described in sera from SLE patients, but they did
not gain much attention before 1972 when Sharp et al. presented their concept of MCTD as a distinct
CTD [1]. Biochemical analyses have shown that the anti-RNP antibodies bind three U1-specific proteins
 R. Gunnarsson et al. / Best Practice & Research Clinical Rheumatology 30 (2016) 95e111 99

A
SR

70 kDa

C Sm

Fig. 2. The U1 ribonucleoprotein complex.

Adapted from Greidinger et al., Rheumatic Diseases Clinics of North America, 2005.

A, C, and 70 kDa in the macromolecular U1esnRNP complex [34e36]. (Fig. 2) This complex contains
many other antigens targeted by CTD-associated autoantibodies, including anti-Smith (Sm). An
optimal protein purification was therefore critical for the diagnostic performance of anti-RNP anti-
bodies. This has become less of a problem with the current enzyme-linked immunosorbent assays
(ELISAs) as mixtures of recombinant proteins A, C, and 70 kDa are predominantly used as the antigen
source.
The available data indicate that the 70-kDa antigen is the main target of serum anti-RNP antibodies
in MCTD [34,35]. It should, however, be noted that the relative frequencies of the circulating anti-U1
protein A, anti-U1 C, and anti-U1 70 kDa antibodies have not been widely assessed, either in MCTD
or in any other CTD marked by anti-RNP antibodies.
The role of the anti-RNP antibodies in the pathogenesis of MCTD remains unknown, but recent data
have reinforced the notion that the autoantibody production may be genetically determined and
driven by distinct subsets of HLA-restricted T cells. Recently, we compared the HLA profiles, deter-
mined by sequence-based typing of HLA-B* and DRB1* in four groups of CTD patients of Norwegian
ancestry: MCTD (n ¼ 155), SLE (n ¼ 96), SSc (n ¼ 95), PM/DM (n ¼ 84), and a control group of healthy
individuals (n ¼ 282). The HLA DRB1*04:01 (OR 2.82; p ¼ 3.64  108) was confirmed to be a major risk
allele for MCTD, in addition to HLA-B*08 (OR 2.05; p ¼ 1.31  104), while DRB1*04:04, DRB1*13:01,
and DRB1*13:02 were protective [37]. This was to date the largest HLA study on MCTD, and results
obtained herein are similar to those in the previous, much smaller studies [38e40]. Very recently, a
study from Poland confirmed HLA DRB1*04:01 as a risk allele for MCTD [41]. A large genetics study
conducted in the UK on juvenile dermatomyositis (JDM) and JDMescleroderma (JDMeSSc) overlap
showed that all anti-U1-RNP antibody-positive patients (6% of the total patient cohort) had at least one
copy of HLA-DRB1*04, thus probably reflecting that the HLAeDR4 association observed in adult MCTD
is also present in children [42]. Whether the observed HLA DRB1*04:01 association is primarily linked
to anti-RNP or MCTD is of major concern. If it were linked to anti-RNP, one would expect to see the
same HLAeDR4 association in anti-RNP-positive SLE or SSc. Unfortunately, the data on this issue are
conflicting; Hoffman et al. [43] and Smolen et al. [44] reported a clear association between anti-RNP
and HLAeDR4 in SLE, whereas Olsen et al. [45] did not find any such association.
Data from HLA transgenic mice with MCTD-like disease suggested that the anti-RNP antibodies
could be pathogenic, but no human data are available on this issue [46]. To our knowledge, no studies
are available on the pathogenic role of anti-RNP in other CTDs.

Clinical features

Raynaud's phenomenon and “puffy hands”

Raynaud's phenomenon (RP) was first described by Dr. Maurice Raynaud in 1862, which occurs in a
primary and secondary form [47]. Secondary RP, associated with the underlying chemical and/or
100 R. Gunnarsson et al. / Best Practice & Research Clinical Rheumatology 30 (2016) 95e111

structural changes in the vessel walls, is seen in all CTDs, but it is most prevalent in SSc and MCTD [16].
It has been reported that patients with secondary RP due to SSc, MCTD, and DM have characteristic
structural changes in their nailfold capillaries [48e52]. Unfortunately, the nailfold capillaroscopy
patterns of the different CTDs have not been systematically compared. No nailfold capillaroscopy data
are currently available from the Norwegian cohort, but capillaroscopy data from the Hungarian MCTD
cohort showed that 38% had a scleroderma capillaroscopy pattern [53]. In the nationwide Norwegian
cohort [13], RP was reported in 99% of the patients, comparable with the previous studies [8,10,17].
Swollen or “puffy” hands are among the most common disease manifestations reported in adult and
juvenile MCTD cohorts (Table 2). The mechanisms behind the symmetrical hand swelling are not clear,
but tenosynovitis and/or endothelial cell dysfunction have been suggested [54]. Adult MCTD cohort
studies have reported “puffy hands” in 60e94% of the patients [10,17,55].

Myositis
Myositis is included as one of the major criteria in all the four criteria sets for MCTD (Table 1), but
the actual data on muscle involvement are quite limited [56]. The available data indicate that 35e79%
of the MCTD patients will develop signs of myositis during the course of the disease [1,5,8,10,13,17,55],
but myositis is rarely present at disease onset [10]. It appears that the MCTD-associated muscle
involvement has the same distribution as PM/DM, but seems to cause less permanent damage [57].
Data on muscle histology in MCTD are limited and there are no studies on the potential role of muscle
magnetic resonance imaging (MRI).
In a large European primary myositis cohort [58], autoantibodies to anti-U1 snRNP antibodies were
reported in 6% (25/417). The anti-RNP-positive patients were treated with a lower steroid dose
compared to the patients with classical PM or DM and succeeded in reducing it earlier and seemed to
have a better prognosis regarding muscle involvement. Whether any of the anti-RNP-positive patients
were diagnosed with MCTD has not been reported.

Table 2
Accumulated frequencies of clinical features in adult- and juvenile-onset MCTD.

Clinical features Adult onset %a Juvenile onset %b

Raynaud phenomenon 99 93e100

Puffy hands 93 61e91
Anti-RNP positive 100 93e100
SLE-like manifestations
Arthritis 79 78e97
Facial erythema 41 9e69
Leukopenia 31 30e50
Thrombocytopenia 12 6e21
Pleuritis 14 10e21
Pericarditis 12 16e28
Lymphadenopathy N/A 21e43
SSc-like manifestations
Sclerodactyly 34c 26e86
Pulmonary fibrosis on CT 35 25e30
Hypomotility or dilatation of esophagus 50 7e21
PM-like manifestations
Muscle weakness 33 29e70
Elevated CK 33 35e68
Myogenic pattern in EMG N/A 25e38
Other manifestations
Pulmonary arterial hypertension 4 9
Nephritis 3 6e21
Anemia 27 64
a
Reported frequencies on adult onset disease are from the population based Norwegian, nation-wide MCTD cohort study
[13,33,143].
b
Reported frequencies in juvenile onset disease are range with percentages from four different cohort studies
[12,14,140,151].
c
Sclerodactyly was reported as distal to PIP (proximal interphalangeal) joints.
 R. Gunnarsson et al. / Best Practice & Research Clinical Rheumatology 30 (2016) 95e111 101

Arthritis and arthralgia

Joint symptoms are commonly associated with MCTD in most studies [59], and arthritis is included
in all the criteria sets (Table 1). The most common pattern appears to be polyarthritis, but the extent of
joint damage caused remains unclear. In a study dating back to 1977, erosive lesions were described in
as many as 53% (9/17) of the MCTD cases, but the representative nature of this study is not clear. Re-
ports on the use of MRI and ultrasound are emerging, but no systematic data have been published
[60,61].

Interstitial lung disease

Several small, single-center reports have indicated that ILD is one of the most severe disease
complications in MCTD [8,10,18,21,22,62,63]. Cross-sectional data from the Norwegian nationwide
study showed that 52% of the patients examined (n ¼ 126) had abnormal findings on high-resolution
computed tomography (HRCT) of the lungs, most commonly reticular patterns consistent with lung
fibrosis (35%). Fibrosis was quantified as minor in 7%, moderate in 9%, and severe in 19% of the patients.
Fibrosis was uniformly concentrated in the lower parts of the lungs and was not associated with
smoking. Patients with severe lung fibrosis had lower pulmonary function test (PFT) values, shorter 6-
min walk distance, a higher mean NYHA (New York Heart Association Functional Classification)
functional class, and increased mortality (see section on prognosis). A newly published retrospective
cohort study of 39 MCTD patients compared baseline and follow-up PFT and HRCT data over a 10-year
period [64]. At baseline, 51% of the patients had abnormal PFTs. Forced vital capacity (FVC) was slightly
reduced at baseline (77% of predicted) but remained stable after 10 years. The diffusion capacity for
carbon monoxide (DLCO) decreased from 84% to 71%, and the median lower lobe ILDeHRCT score
progressed from 7.5% at baseline to 11.2% at follow-up [64]. Few studies have investigated ILD in JMCTD,
but a HRCT-based study showed that discrete ILD changes were present in 25% (6/24) patients, possibly
indicating that JMCTD patients have less severe lung involvement than adult-onset MCTD [62].

Pulmonary hypertension
Precapillary PH is an important cause of morbidity and mortality in CTD, particularly in SSc [65].
According to the 2015 European Society of Cardiology (ESC) and the European Respiratory Society (ERS)
[66], pre-capillary PH is defined as a mean pulmonary arterial pressure (mPAP) of 25 mm Hg at rest by
right-sided heart catheterization (RHC), coinciding with a pulmonary artery wedge pressure (PAWP)
within normal limits of 15 mm Hg. According to the 2013 updated clinical classification of PH [67],
patients with CTD-related precapillary PH belong to two main categories. They have either isolated
pulmonary arterial hypertension (PAH; Group 1.4.1) or PH-associated with ILD (PHeILD; Group 3.2).
Data on PH in MCTD are limited. The national PAH registry in the UK (with a population of about 60
million) [68] included 36 MCTD patients with PH, thus indicating a total MCTDePH prevalence of 0.6
per million.
Three earlier small single-center cohort follow-up studies estimated the total frequency of PH in
adult MCTD at 23e24% [7,8,10]. PH seems to be frequent (6e9%) in single-center JMCTD cohorts [12,14].
In the Norwegian cohort study, all the MCTD patients were screened at study enrollment by echo-
cardiography and patients suspected to have PH were referred for RHC. After enrollment, the patients
were followed up for a mean period of 5.6 years. At baseline, 2.0% of the patients (3/147) had an
established PH. Two additional PH patients were identified during follow-up, thereby yielding a total
PH frequency in the cohort of 3.4% (5/147). Two of these patients had PAH and three had PHeILD. A
recently published study from Hungary reported that 18% of the MCTD patients (50/280) developed
PAH after a mean follow-up time of 14.5 years [53]. There are several possible reasons for the much
higher PH frequency observed in this study, the most obvious being the difference in follow-up time.
Another possible difference is the patient selection: the Norwegian study was population based, while
the Hungarian cohort was derived from a large CTD center [13,53].
There are no published CTDePAH epidemiological studies conducted in Asia, but recent data from a
single-center Japanese cohort study of 70 patients with PAH associated with CTDs (PAHeCTD) [69]
showed that among CTDs, MCTD was most commonly associated with PAH (43%, 30/70), followed
by SLE (29%, 20/70) and SSc (19%, 13/70). This is in contrast with the studies from Europe and USA,
where the majority of patients with PAHeCTD have SSc. For example, in the national UK registry study
102 R. Gunnarsson et al. / Best Practice & Research Clinical Rheumatology 30 (2016) 95e111

referred earlier, 74% (315/429) of the CTDePAH patients had SSc, 8% MCTD (36/429), and 8% SLE (35/
429) [68]. In the US-based REVEAL prospective PAH Registry (Registry to Evaluate Early and Long-Term
PAH Management), the majority of the CTD PAH patients had SSc (500/804; 62%), while 16% had SLE
and 9% had MCTD [70].

Skin manifestations and alopecia

Skin involvement is included in the Sharp and Kasukawa criteria sets (Table 1) [27,28]. There is a
lack of newer data on rash in MCTD, but the long-term study by Burdt et al. described erythematous
skin rash at onset in 13% and cumulatively in 53% [10]. A Hungarian cohort study of Hajas et al. reported
that a cumulative 36% of the patients developed some sort of skin involvement in the course of the
disease, including photosensitivity, malar rash, telangiectasia, or hypo- and hyperpigmentation [53]. In
the study of Sullivan and coworkers, 29% had malar rash and 41% had alopecia [8]. Interestingly, a small
pathology study including eight patients with MCTD showed that the epidermal pathology mimicked
that of subacute cutaneous lupus erythematosus (SCLE), but a concomitant vasculopathy parallel to
that observed in DM distinguished it from SCLE [71].

Gastrointestinal involvement
The available data indicate that esophageal dysmotility and hypomotility are common in MCTD
[13,53,72,73], and these findings are included in the Sharp and Kasukawa criteria sets [27,28] but not in
the other two (Table 1). There are isolated reports of MCTD with autoimmune hepatitis [74e76], pri-
mary biliary cirrhosis [77], portal hypertension [78], and protein-losing enteropathy [79,80]; however,
overall, it appears that GI (gastrointestinal) complications other than dysmotility of the esophagus are
infrequent in MCTD. Interestingly, angiodysplasia of the gastric antrum, a recognized complication of
SSc [81], has only been reported in a single MCTD patient [82].

Hematological manifestations
There are no recent data on the hematological alterations in MCTD, but the older studies indicate
that anemia and leucopenia are common during the disease course whereas thrombocytopenia is
reported to be infrequent [10,13,17,53].

Neurological manifestations
Trigeminal neuropathy is the only neurological complication included in any of the criteria sets and
probably the most common nervous system problem encountered in the disease [6]. Neuropsychiatric
manifestations that are commonly observed in SLE and other central nervous complications appear to
be very rare in MCTD [83]. A recent study from Hungary showed that the frequency of sensory-neural
hearing loss, as registered by audiograms, was twice as high in patients with MCTD compared with
healthy controls [84]. This potentially important finding has not been reproduced in other cohorts.

Renal involvement
Renal involvement is not included in any of the four criteria of MCTD, but data accumulated from
several studies indicate that up to one-fifth (20%) of the MCTD patients eventually will develop some
form of renal disease, most commonly, an immune complex nephropathy histologically classified as
membranous glomerulonephritis (GN) [55,85]. It does, however, seem that the kidney involvement in
MCTD is mostly subclinical, with a good prognosis. In the Norwegian nation-wide cohort, <3% had
minor kidney involvement during the course of the disease (unpublished data). In the Hungarian
cohort, 4% had kidney involvement, with 27% (3/11) of those having thrombotic thrombocytopenic
purpura-associated nephropathy and the remaining 73% (8/11) with GN, mostly ISN/RPS 2003 class II
mesangial GN [13,86].

Relationship with other connective tissue diseases

Recent studies have confirmed that the HLA profiles of MCTD are distinctly different from the HLA
profiles of the ethnically matched healthy controls and the HLA profiles of SLE, SSc, and PM/DM (see
 R. Gunnarsson et al. / Best Practice & Research Clinical Rheumatology 30 (2016) 95e111 103

Update on the immunological and clinical features of MCTD section on immunological features for
details). This genetic finding supports that MCTD is a disease entity distinct from SLE, SSc, and PM/DM.
There are, however, no functional data on how the HLA*B08 and DRB1*04:01 alleles associated with
MCTD may contribute to the formation of anti-RNP autoantibodies or disease pathogenesis. The
general scenario for CTD development, where an individual with a predisposing genetic background
must be exposed to the environmental risk factors to develop systemic, chronic immune activation, is
also most likely relevant for MCTD, but there are no data on potential environmental risk factors.
At a more clinical level, it would be interesting to know more about the phenotype, and phenotypic
stability of MCTD in relation to immunological and genetic profiles. The data published on the short-
term and long-term phenotypic stability of MCTD are conflicting, partly due to differences in disease
definitions. The studies on phenotypic stability fall into two main categories: study with a follow-up of
patients with anti-RNP antibodies [6,17,87,88] and the other with a follow-up of patients with a clinical
MCTD diagnosis [10,17,87,89]. The conclusions from these studies have been mixed, with some indi-
cating that MCTD evolves into another CTD in the majority or even in all patients [6,88], whereas others
find that MCTD in most patients does not evolve into other CTDs [10,17,89]. It is, however, important to
note that phenotypic stability has never been evaluated in population-based, unselected MCTD co-
horts. The lack of systematic data on the fluctuations and changes of immunological and clinical fea-
tures over time in CTD in general is of concern. In particular, whether temporal changes in individual
patients may lead to the development of a new CTD (“clinical shift”) or gradually change the CTD
phenotype and/or disease activity pattern over time (“clinical drift”) is not known.
We believe that the issues of clinical shifts and drifts are of particular relevance in prospective
studies on MCTD and also in cohorts comprising patients who do not fulfill any classification criteria
and therefore are classified as undifferentiated CTD (UCTD). Clinical experiences indicate that, over
time, some UCTD patients develop distinct CTDs such as MCTD, SLE, or DM/PM [27e30,90e93], but
their clinical and immunological profiles have not been systematically assessed. Another important
issue in the clinic is the challenge of delineating MCTD from other CTDs, particularly SLE and SSc. This
can be attributed partly to the complex nature of the disease phenotype and to the challenges in
working with criteria sets of MCTD. All the four criteria sets of MCTD were defined as diagnostic (and/
or classification) criteria and all were based on the same principle to count accumulated clinical and
immunological features. This principle is used in most CTD classification criteria, including the current
SLE and SSc criteria [93e97]. The principle of counting accumulated features is complicated in asso-
ciation with the concept of phenotypic stability. A “classical” MCTD patient with ANA (antinuclear
antibodies), high-titer anti-RNP, RP, “puffy hands,” leukopenia, polyarthritis, myositis, and low-grade
ILD evaluated by HRCT can be considered as an example. This patient has met the four criteria sets
for MCTD. However, following nailfold capillaroscopy, the patient is found to have a typical “sclero-
derma pattern” [97] and has also met the 2013 ACR/EULAR classification criteria for SSc [96]. Does this
finding imply phenotypic instability, and does it merit reclassification from MCTD to SSc? If this patient
develops membranous GN, he/she would also meet the classification criteria for SLE. Should the GN
then be regarded as an additional feature of the MCTD, or signify that the patient has “drifted” to SLE?
In some cases, the observed clinical shift may have therapeutic consequences. An obvious example
is the patient with MCTD who suddenly develops nephritis and has anti-double-stranded DNA anti-
bodies and low complement levels (C3 and/or C4). Most clinicians would probably agree that it would
be correct to change the diagnosis from MCTD to SLE and start treatment for lupus nephritis. The
conditions with the disease onset marked by leukopenia, rash, arthritis, ANA, and high anti-RNP
antibody titers and a disease course with development of ILD, myositis, and worsening RP are less
obvious. We believe that most clinicians would initially diagnose this patient with SLE, but we are less
convinced that the same clinicians would regard this case as a “phenotypic shift” and change the
diagnosis to MCTD.

Treatment and advances in therapy

The initial assumption by Sharp and coworkers that MCTD was responsive to corticosteroid therapy
and yielded a favorable outcome has not been well established [1]. No randomized controlled trials of
104 R. Gunnarsson et al. / Best Practice & Research Clinical Rheumatology 30 (2016) 95e111

therapies for MCTD have been performed. Instead, therapy is commonly directed by the clinical
manifestations and is based on knowledge and experience from treating other related CTDs.
Symptomatic treatment of RP, the most common symptom of MCTD, consists of general advice
(keeping warm, and avoiding injury, smoking, and caffeine) [98]. Treatment with oral calcium channel
blockers that block voltage-gated calcium channels (VGCCs) in cardiac muscle and blood vessels de-
creases total peripheral resistance and increases the peripheral blood flow with effects on RP [99].
Nifedipine is commonly used, but other dihydropyridine calcium channel blockers can be used, such as
amlodipine, felodipine, isradipine, or others. Administration of intravenous prostaglandin, most often
iloprost, has documented effects on RP [98]. Some case reports document a positive effect from topical
nitroglycerin on RP, but for all other interventions, the current evidence is limited, conflicting, or
nonexistent [47,98].
The treatment of digital ischemic ulcers is challenging. The study of SSc shows that prostaglandin
infusions [100], sildenafil [101,102], and digital sympathectomy [103,104] have effects in the acute
phase. In addition, it appears that subcutaneous, local doses of botulinum toxin A may be efficacious
[105,106]. Oral use of the endothelin 1 receptor inhibitor, bosentan, reduces the risk of new digital
ulcers in SSc [107e109], but similar to the other drugs, it has not been evaluated in MCTD.
Several researchers consider anti-malarial drugs, most commonly hydroxychloroquine (HCQ), to be
the basic immune-modulating treatment for MCTD; however, in contrast to SLE, no data support this
notion. In cases with pleuritis, pericarditis, arthritis, myositis and/or ILD, corticosteroids, either orally
or intravenously, are still the cornerstone of therapy, mostly in combinations with other treatments.
Steroid-sparing treatments for MCTD, commonly used as second-line therapy, include oral or intra-
muscular methotrexate, cyclosporine, azathioprine (AZA), and mycophenolate mofetil (MMF). Severe
ILD is also treated with intravenous cyclophosphamide.
There are no specific guidelines in the treatment of PH associated with MCTD, and the treatment
options are derived from studies of PAH-specific therapies in idiopathic PAH and PAH associated with
SSc. Calcium channel blockers should be used for treatment of the few patients with PAH who
respond to a vasodilator challenge at RHC (<5%) [66]. Patients with CTD-related PAH (CTDePAH) are
sometimes treated with anticoagulation, mostly warfarin, to reduce the risk of intrapulmonary
thrombosis and thromboembolism, but the efficacy of this treatment is yet to be determined
[66,110,111]. Notably, patients with CTDePAH, including SScePAH, have a higher rate of bleeding than
those with an idiopathic PAH [110,112,113]. Prospective studies with a subset analysis are required to
accurately assess whether increased bleeding risk offsets the apparent benefit of anticoagulation in
these patient groups.
PAH can be more specifically treated with endothelin receptor antagonists (bosentan, ambrisentan,
or macitentan), phosphodiesterase 5 inhibitors (sildenafil, tadalafil, or vardenafil), and prostanoids
(epoprostenol, treprostinil, or inhaled iloprost) [66,114e123]. In treatment-resistant cases, a combi-
nation of two and even three of these treatment options can be used. The most preferred therapeutic
option is an oral treatment with endothelin receptor antagonist in combination with phosphodies-
terase 5 inhibitors [124]. Intravenous, inhaled, or subcutaneous administration of prostanoid treatment
has several and significant disadvantages [66]. Recent reports indicate that selexipag, an oral, selective
prostacyclin receptor, shows promise [125]. A recently published, phase III randomized double-blind
prospective trial among 1156 patients with PAH included treatment of naïve patients and those
receiving a stable dose of an endothelin receptor antagonist, a phosphodiesterase type 5 inhibitor, or
both. The risk of the primary composite end point of death or a complication related to PAH was
significantly lower with selexipag [125e127]. Finally, in contrast to SSc, there are reports of MCTD
patients with PAH who have responded to an immunosuppressive treatment alone [128].
Currently, there is little experience in the use of “biological disease-modifying anti-rheumatic
drugs” in MCTD. There are a few reports of both adult [129e134] and juvenile [135] MCTD cases treated
with B cell depletion therapy with rituximab with success, mainly for treatment-resistant arthritis,
myositis, and thrombocytopenia. There are only very few reports of MCTD cases treated with TNF
(tumor necrosis factor) alpha inhibitors [136e138], possibly because the initial reports indicated severe
adverse effects. We have, nevertheless, in our own hospital practice, a few MCTD patients with
treatment-resistant arthritis who have been successfully treated with anti-TNF alpha treatment (un-
published data).
 R. Gunnarsson et al. / Best Practice & Research Clinical Rheumatology 30 (2016) 95e111 105

Prognosis and prognostic features

Outcome

Outcome assessments in MCTD are complex. In previous sections, we discussed outcomes in

relation to phenotypic stability. In this section, we focus on more general outcome measures, including
survival and organ damage. Of note, there are no uniform standardized outcome measures or damage
measures for MCTD.

Mortality

Current knowledge about the mortality of patients with MCTD is based on a handful of single-
center follow-up studies with small cohorts consisting of 15e47 patients, with a mean observation
time between 5 and 17 years, in addition to two larger cohort studies with 147 and 280 patients.
Overall, 10 studies [5,6,8,10,13,53,139e143] represent a total of 644 MCTD patients of whom 63 had
JMCTD. These studies covered a total of 77 deaths. Five studies [6,8,10,53,141] with a time span
greater than a mean of 10 years (11e17 years) of follow-up included totally 406 patients and reported
50 deaths that cumulatively yielded a crude mortality rate of 12%, varying between 8 and 36%
[6,8,10,53,141]. The causes of death in these studies were mainly related to PAH/PH and/or ILD in
addition to infections and malignancies. In JMCTD, the mortality rates have been reported to be lower,
varying between 2.8 and 7.9%, according to a nationwide Japanese study and a literature review by
Michels [14,140].
It is difficult to draw meaningful conclusions from these studies because of the heterogeneity of
the samples, different follow-up times, and the fact that there were no comparisons with the
matched controls from the general population. Clearly, further studies on mortality in MCTD are
warranted.

Organ damage (PH and ILD)

PH was a major cause of mortality in the Norwegian nationwide cohort, responsible for a total of
25% (3/12) of all deaths, after a mean follow-up of 5.6 years [13,33,143]. In a Hungarian regional cohort
study, a total of 50 patients had developed PAH, after a mean of 14.5 years (±3.7 years) following the
diagnosis. In addition, PAH was reported to be the major cause of mortality, affecting 41% (9/22) of the
deaths in the cohort [53].
Despite interobserver variability, the World Health Organization functional class (WHOeFC) re-
mains one of the most powerful predictors of survival at both diagnosis and follow-up of PH
[66,144e146]. A recently published study from the US REVEAL Registry showed a markedly lower 3-
year survival of patients with newly diagnosed SSc-associated PAH (51.2% ± 4.0%, n ¼ 166) when
compared with those with newly diagnosed other (non-SSc) CTD-associated PAH, probably including
MCTD cases (76.4% ± 4.6%, n ¼ 88) [70]. A very recent study from a large prospective PH database in the
UK aimed to study the clinical and hemodynamic characteristics and survival of CTDePAH patients. The
study identified a total of 342 CTDePAH patients, with 36 (11%) being anti-U1RNP antibody-positive
patients [147]. Patients with anti-U1RNP antibodies were younger and less functionally impaired than
the anti-U1RNP-negative patients. In multivariable analysis, anti-U1RNP positivity was associated with
decreased mortality.
ILD seems to be common in MCTD [8,10,18,21,22,33,53,62,63], and it affects the pulmonary function,
overall physical capacity, and mortality [33]. Currently, there are few reliable long-term follow-up data
on ILD in MCTD and insufficient data on the prognostic factors for evolving ILD and progression of ILD
[64]. After a mean of 4.2 years of follow-up in the Norwegian nationwide cohort, the mortality in
patients with normal HRCT was 3.3%, compared to 20.8% in patients with severe lung fibrosis (p < 0.01)
[33]. Very recently, we found that anti-Ro52 antibodies may serve as a potential marker for lung
fibrosis in MCTD, but this need to be confirmed in further studies [148].
106 R. Gunnarsson et al. / Best Practice & Research Clinical Rheumatology 30 (2016) 95e111

Clinical implications and future perspectives

Clinical implications

Accumulated evidence from the recent studies indicates that the prevalence and severity of ILD
in MCTD was higher than previously expected. Therefore, we believe that systematic screening for
lung involvement in patients with recent-onset MCTD is warranted. Early diagnosis and
treatment may hopefully delay or stop the progression of lung fibrosis and thereby improve
outcomes.
The prevalence of PH in MCTD, at least in Europe and USA, seems to be lower than expected from
several earlier small cohort studies [7,8,10] and lower than in SSc [143]. There are currently no
guidelines for screening for PH in MCTD such as there are for SSc [149]. Overall, the available evidence
indicates that PAH and PH are less frequent in MCTD in Europe and USA than previously assumed. The
current challenge is if and how we should screen for PH in our MCTD patients. Currently, there are no
guidelines or consensus reports. The evidence-based DETECT algorithm developed for SScePAH may be
used in selecting patients with MCTD for further echocardiography and subsequently RHC, but
currently this protocol has not been formally tested in MCTD [150]. All patients in the Norwegian
cohort with PH (5/5) had elevated serum levels of N-Terminal-pro brain natriuretic peptide (NT-
proBNP) during study inclusion or PH diagnosis, indicating that this marker may be useful as an
additional screening test in MCTD patients with suspected PH [143].

Future perspectives

Little is still known about MCTD. Prospective, population-based, long-term observational studies on
incident MCTD cases will be important for obtaining unbiased data on clinical features, prognosis,
prognostic factors, phenotypic stability as well as reliable estimates on incidence and prevalence.
Larger, long-term studies should also provide novel data on mortality and relative cancer risk matched
to standardized populations. This type of study combined with repeated biological sampling would be
ideal to fill large gaps in knowledge of the distinct immunology, functional genetics, and epigenetics of
MCTD.
However, larger, collaborative, and interdisciplinary efforts should be made to address some of the
more complex questions related to anti-RNP and the clinical disease spectrum associated with these
antibody specificities. Three of these questions, and our proposed approach to them, are outlined in
Box 1.

Box 1
Key research questions related to the anti-RNP-associated CTDs.

Q1; Is the concept of MCTD as a separate disease entity meaningful in terms of prognosis and
management?
Could possibly be answered by a large, international multicenter study aiming to define a new
set of classification criteria for MCTD. This study would have to include a large number of anti-
RNP-positive patients with CTDs other than MCTD.
Q2; Is there a systematic difference in the fine specificity of the anti-RNP response across the
various CTD entities?
Could possibly be answered by analyses of biological material (plasma and B lineage cells)
from the same cohort used to approach Q1.
Q3; Is there a systematic correlation between genotype and phenotype in the anti-RNP-positive
clinical disease spectrum?
Could be answered by genetic analyses of population-based cohorts that should include all
patients with anti-RNP-positive disease in the defined study area.
 R. Gunnarsson et al. / Best Practice & Research Clinical Rheumatology 30 (2016) 95e111 107

Future immunology, genetic, epigenetic and pathology studies will provide new diagnostic and
prognostic tools that will most certainly change our current view of MCTD, as well as the common-
alities and differences with other CTDs.

Conflict of interest

The authors report that they have no conflicts of interest.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.berh.2016.03.

002.

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