An Observational Cross-Sectional Study of Varied Clinical Manifestations of Connective Tissue Disorders and their Association with Antinuclear Antibodies in a Tertiary Care Center.

CONTEXT: Connective tissue disorders (CTD) occur in 3-5% of the population. The advent of antibodies to extractable nuclear antigens (ENA) has become a reliable predictor to establish the diagnosis of CTD, subclassify patients into prognostic groups, and monitor disease activity. AIMS: The aim of this study was to (a) study the frequency of cutaneous manifestations, systemic manifestations, and anti-ENA antibodies in CTD; (b) determine the association between systemic manifestations and ENAs; and (c) determine the association between cutaneous and systemic manifestations of CTD. SUBJECTS AND MATERIALS: An observational cross- sectional study was conducted on 50 patients diagnosed to have CTD. The clinical profile and antibodies to ENA (ANA Profile) reports were retrieved and studied.
RESULTS: The major dermatological manifestations were skin tightness (36%), salt and pepper pigmentation (30%), Raynaud's phenomenon (28%), and malar rash (28%). The common antibodies seen were anti SS-A (36%), anti-UI-ribonucleoprotein (U1-RNP) (34%), anti-dsDNA (32%), and anti-Sm (24%). Patients with anti-Sm and anti-dsDNA antibodies had increased frequency of renal manifestations. A strong association with significant P values was seen between neurological manifestations and anti-Sm antibody, and cardiovascular manifestations and anti-RNP antibody. An association between gastrointestinal manifestations and malar rash as well as neurological manifestations and photosensitivity was also seen.
CONCLUSIONS: ENA panel predicts systemic involvement, thus helping in the multidisciplinary management. Cutaneous manifestations of CTD can be an early predictor in giving a clue to impending systemic manifestations.

Introduction

Connective tissue disorders (CTD) are multisystem diseases with conspicuous mucocutaneous manifestations. These include systemic lupus erythematosus (SLE), scleroderma, dermatomyositis, mixed connective tissue disorder (MCTD), Sjogren's syndrome, and overlap syndrome/undifferentiated connective tissue disorder (UCTD). They present with specific signs, nonspecific signs, or with an overlap of manifestations, which makes their diagnosis challenging.

Antinuclear antibodies (ANA) are a specific class of autoantibodies that have the capacity to adhere to and destroy certain structures inside the cell nucleus.[1] Extractable nuclear antigens (ENA)/Soluble cellular antigens are a heterogeneous group of ribonuclear proteins and non-histone proteins with different functions in nuclear metabolism.[2] ENAs are detected by counterimmunoelectrophoresis, Western blotting, and enzyme-linked immunosorbent assay.[3]

The common indications of ENA analysis are to establish the diagnosis of CTDs in patients with suggestive clinical features, to exclude CTDs in patients with few or uncertain clinical findings, to subclassify patients known to have CTDs into prognostic groups, and to monitor disease activity.[3] The role of certain antibodies to ENAs as diagnostic and prognostic markers indicating underlying organ damage is well known. The rationale behind this study was to explore new associations between the clinical (cutaneous and systemic) and ENA profile in CTDs.

The aims and objectives were to study the frequency of cutaneous manifestations, systemic manifestations, and anti-ENA antibodies in CTD, and to determine the association between cutaneous and systemic manifestations of CTD as well as association between systemic manifestations and antibodies to ENA.

Subjects and Methods

We conducted an observational cross-sectional study in a tertiary care center in Mangalore, South India. Fifty cases diagnosed to have CTDs from the inpatient records of department of dermatology from June 2016 to May 2017 were included. Institutional ethical clearance and waiver's consent were obtained. A detailed history was taken and clinical examination was carried out. Blood investigations, namely, complete blood count, erythrocyte sedimentation rate, blood sugar, renal function test, liver function test, urine routine and microscopic examination, 24 h urine protein levels, ANA by immunofluorescence, ENA by immunoblot technique, serum complement levels, ECG, X-ray chest, X-ray of joints, and abdominal sonography, were done. Additional investigations like pulmonary function test, barium swallow, endoscopy, echocardiography, high resolution computed tomography (HRCT) of thorax, electroencephalogram (EEG), and MRI were done in selected cases. Referrals were given to internal medicine department for detailed systemic evaluation. The diagnosis of various CTDs was made as per various guidelines and criteria recommended by Rheumatology society (2012 SLICC criteria for SLE, 2013 ACR/EULAR criteria for scleroderma, Bohan and Peter criteria for dermatomyositis, Alarcon-Segovia and Kahn criteria/Kasukawa's criteria for MCTD, etc.). Clinical profile and antibodies to ENA (ANA profile) reports were retrieved and studied. Statistical analysis was done using SPSS software version 22.0. Chi-square test was done to study the association between two variables. P values of less than 0.05 were considered statistically significant.

Results

Epidemiology

In the current study, the mean age at presentation was 38.2 ± 14.5 years. The youngest age of presentation was 14 years and the eldest was 71 years. There were 43 (86%) females and 7 (14%) males.

Diagnosis

Out of 50 cases, 20 (40%) had SLE and 12 (24%) had diffuse cutaneous systemic sclerosis, 6 (12%) had overlap syndromes/UCTD, 5 (10%) had MCTD, and 3 (6%) had limited cutaneous systemic sclerosis. Interestingly, we had two cases of juvenile dermatomyositis, one case of adult dermatomyositis and one case of Sjogren's syndrome.

Cutaneous and systemic manifestations

Cutaneous manifestations were present in 47 (94%) cases. The various cutaneous manifestations seen are given in Table 1 with skin tightness (36%) being the commonest presentation. The systemic involvement is summarized in Table 2.

Table 1

Cutaneous manifestations in CTD

Cutaneous manifestations	Number of patients (percentage)
Salt and pepper pigmentary changes	15 (30)
Malar rash	14 (28)
Skin tightness	18 (36)
Sclerodactyly	11 (22)
Pitted scars	11 (22)
Puffy fingers	3 (6)
Heliotrope rash	3 (6)
Gottron’s papules	2 (4)
Oral ulcers	10 (20)
Calcinosis cutis	1 (2)
Raynaud’s phenomenon	14 (28)
Photosensitivity	15 (30)
Palpable purpura	1 (2)

Table 2

Systemic manifestations in CTD

Systemic manifestations	Number of patients (percentage)
Gastrointestinal (GIT)	25 (50)
Neurological (CNS)	3 (6)
Respiratory	17 (34)
Renal	12 (24)
Cardiovascular (CVS)	7 (14)
Musculoskeletal (MSK)	24 (48)

Antibodies to ENA

A total of 47 (94%) patients had a positive ANA profile. The various antibodies to ENA seen were SS-A (36%), U1-RNP (34%), dsDNA (32%), Sm (24%), Scl-70 (22%), SS-B (18%), centromere protein B (CENP-B) (16%), and others like Ro-52, histones, and Jo-1 in small proportions. Thirty-three patients (66%) showed positivity for more than one autoantibody.

Association between systemic manifestations and anti-ENA

Gastrointestinal involvement was significantly associated with CENP-B antibody. Presence of anti-Sm and anti-dsDNA antibodies was significantly associated with renal involvement. Anti-Sm antibody and anti-RNP antibody were significantly associated with neurological and cardiovascular manifestations, respectively [Table 3].

Table 3

Association between systemic manifestations and antibodies to ENA

Antibodies to ENA		Gastrointestinal involvement				χ2	P
		Absent (n=25)		Present (n=25)
		Count	Percent	Count	Percent
CENP-B	Absent	24	57.1	18	42.9	5.36*	0.021
	Present	1	12.5	7	87.5
		Renal involvement
		Absent (n=38)		Present (n=12)
		Count	Percent	Count	Percent
Sm	Absent	33	86.8	5	13.2	10.2**	0.001
	Present	5	41.7	7	58.3
dsDNA	Absent	29	85.3	5	14.7	5.03*	0.025
	Present	9	56.3	7	43.8
		Neurological involvement
		Absent (n=47)		Present (n=3)
		Count	Percent	Count	Percent
Sm	Absent	38	94.7	0	5.3	6.15**	0.006
	Present	9	91.7	3	8.3
		Cardiovascular involvement
		Absent (n=43)		Present (n=7)
		Count	Percent	Count	Percent
U1-RNP	Absent	31	93.9	2	6.1	5.08*	0.024
	Present	12	70.6	5	29.4

*Significant at <0.05 level, **Significant at 0.01 level

Association between systemic manifestations and cutaneous manifestations

Patients with malar rash had an increased frequency of gastrointestinal manifestations. Photosensitivity was more common in patients with neurological manifestations [Table 4].

Table 4

Association between cutaneous and systemic manifestations of CTD

Cutaneous manifestations		Gastrointestinal involvement				χ2	P
		Absent (n=25)		Present (n=25)
		Count	Percent	Count	Percent
Malar rash	Absent	17	42.5	19	57.5	4.5*	0.034
	Present	8	85.7	6	14.3
		Neurological involvement
		Absent (n=47)		Present (n=3)
		Count	Percent	Count	Percent
Photosensitivity	Absent	35	100.0	0	0.0	7.45**	0.006
	Present	12	80.0	3	20.0

*Significant at 0.05 level. **Significant at 0.01 level

Discussion

The demographic and clinical profiles of our patients were comparable to other studies.[45] Akin to the original description of the disease, our study also showed that CTDs primarily affect middle-aged people.[45] The onset of SLE in childhood is associated with an increased severity of manifestation of the disease, higher frequency of organ damage, and reduced survival rates.[6] We encountered a rare case of Rowell syndrome (RS) with lupus erythematosus and erythema multiforme like lesions with antibodies to SS-A. Most of the cases of RS have been reported in middle-aged women, whereas our patient was a 15-year-old adolescent male.[7] Two patients presented at a younger age (<18 years) with neuropsychiatric SLE (anti-Sm, anti-dsDNA, anti SS-A positive) and lupus nephritis (anti-Sm, anti-dsDNA positive), indicating a poor prognosis in young individuals.

The most common CTD was SLE and the least common was Sjogren's syndrome. This spectrum of disorders was similar to other studies.[5891011]

Mucocutaneous manifestations were predominantly seen in our study followed by gastrointestinal and musculoskeletal involvement. A study by Budhrani et al.[8] on 90 patients of CTD also showed the predominance of skin manifestations (98.8%) and polyarthritis (85.5%). The spectrum and frequencies of cutaneous manifestations observed were in accordance with other studies.[51213]

Our study showed lesser renal involvement in patients with systemic sclerosis. Renal involvement is uncommon in Indian patients (6–10%) with systemic sclerosis in contrast to Western countries where 23–40% patients have renal involvement. Thus, racial variations may occur in terms of renal involvement in systemic sclerosis.[1114]

Anti-ENA profile positivity of certain antibodies like Sm, dsDNA, U1-RNP, and Scl-70 are markers of underlying organ damage and clinically relevant disease manifestations.[5815] SLE patients with renal involvement showed the presence of anti-Sm and dsDNA antibodies, which was in accordance with previous studies.[1516] Anti-Sm antibodies are thought to be predictors of silent lupus nephritis.[15] The other risk factors for the development of renal involvement are black race, an early age of diagnosis and presence of anti-dsDNA and anti-La antibodies. These patients need to be closely monitored for the development of renal disease.[1718]

Gastrointestinal involvement was predominantly seen in patients with systemic sclerosis (60%) followed by SLE (20%) which was similar to other studies.[1920] It was associated with the presence of anti CENP-B antibody. Steen et al.[21] showed that CENP-B levels had an association with esophageal stricture, whereas some studies have failed to demonstrate this association.[22]

Our study showed that neurological manifestations of SLE were significantly associated with anti-Sm antibody. Presence of anti-Sm antibodies in the cerebrospinal fluid has been associated with acute confusional state in patients with SLE.[15] An increased anti-Sm IgG titer preceding the onset of neurological manifestations has been documented.[23] However, certain studies have not demonstrated this association.[24] The other risk factors for the development of neurological manifestations are presence of cutaneous vasculitic lesions, thrombocytopenia, low levels of complement C3, C4, and antiphospholipid antibodies. Discoid rash and articular manifestations show a negative correlation with neuropsychiatric SLE.[24] The recognition of adverse predictors of neuropsychiatric disease may play an important role in treatment and prevention.

U1-RNP was significantly associated with cardiovascular involvement, hinting it as a marker for prognosis of pulmonary arterial hypertension (PAH). Anti-U1-RNP antibodies have been associated with PAH in systemic sclerosis, SLE as well as MCTD. MCTD-associated PAH has better prognosis than systemic sclerosis-associated PAH. Presence of anti-U1-RNP antibodies is associated with less severe disease activity and improved survival.[25]

Gastrointestinal manifestations were associated with the presence of malar rash. Neurological manifestations were associated with the presence of photosensitivity. We were unable to retrieve any literature regarding the association between cutaneous and systemic manifestations in CTDs and it remains an unexplored area of interest.

Conclusion

Antibodies to ENAs could be utilized as predictors of organ involvement to determine the prognosis of the same. Dermatological manifestations can be a clue to the onset of systemic disease in addition to anti-ENA studies. There is a need for more studies to determine the association between the cutaneous and systemic manifestations in CTDs.

Limitations

Sample size was small and a larger group is desirable to study the association between clinical manifestations and ENA profile.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.