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U1 В Э70-kd autoantibodypositive mixed connective tissue disease in children. A longitudinal clinical and serologic analysis

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Number 11, November 1993, pp 1599-1602
0 1993, American College of Rheumatology
A Longitudinal Clinical and Serologic Analysis
Objective. To obtain longitudinal data on the
clinical, serologic, and immunogenetic features of children with mixed connective tissue disease (MCTD).
Methods. Eleven children with MCTD were followed up for a mean of 9.8 years. Enzyme-linked
immunosorbent assay and immunoblotting were used to
analyze sera for autoantibodies to small nuclear ribonucleoprotein polypeptides. HLA types were determined in
9 patients, by microcytotoxicity and DNA typing.
Results. All 11 children had anti-U1-70-kd autoantibodies. Six of 9 were positive for HLA-DR2, 4 of 9
for HLA-DR4, and 9 of 9 for either HLA-DR2 or DR4.
Presented in part at the conference Park City 111: Pediatric
Rheumatology Into the ~O’S,Park City, UT, March 1991.
From the Division of Immunology and Rheumatology,
Department of Internal Medicine, and the Departments of Pathology
and Child HealthiPediatrics, University of Missouri-Columbia
School of Medicine, and the Medical Research Service, Department
of Veterans Affairs, Harry S Truman Memorial Veterans Hospital,
Columbia, Missouri.
Dr. Hoffman’s work was supported in part by the Department of Veterans Affairs and by NIH grant AR-41051.
Robert W. Hoffman, DO: Associate Professor, Departments of Internal Medicine and Pathology, University of MissouriColumbia School of Medicine; James T. Cassidy, MD: Professor,
Departments of Child Health and Internal Medicine, University of
Missouri-columbia School of Medicine; Yoshihiko Takeda, MD,
DMS: Research Assistant Professor, Department of Internal Medicine, University of Missouri-Columbia School of Medicine; Elaine
I. Smith-Jones, MSN: Clinical Nurse Specialist, Department of
Internal Medicine, University of Missouri-Columbia School of
Medicine; Grace S. Wang, MS: Laboratory Supervisor, Antinuclear
Antibody Laboratory, Department of Pathology, University of
Missouri-Columbia School of Medicine; Gordon C. Sharp, MD:
Michael Einbender Professor of Internal Medicine and Professor of
Pathology, University of Missouri-Columbia School of Medicine.
Address reprint requests to Robert W. Hoffman, DO,
FACP, Research Service (151), Harry S Truman Memorial Veterans
Hospital, 800 Hospital Drive, Columbia, MO 65201.
Submitted for publication March 9, 1993; accepted in revised form April 26, 1993.
Outcomes were favorable with no functional impairment in 8 of the 11 children and were poor in 3.
Conclusion. The frequency of HLA-DRZDR4 is
increased among children with anti-U1-70-kd autoantibody positive MCTD.
The long-term outcome in children with mixed
connective tissue disease (MCTD) is unclear, with
conflicting reports of both severely adverse as well as
more favorable clinical outcomes described in the
literature (1). Since the publication of earlier reports
on MCTD in children, there have been substantial
advances in the characterization of distinct autoantibodies against individual U small nuclear ribonucleoprotein
([UIsnRNP) polypeptides and in the characterization of
HLA genotypes (2,3). In the present report, we present
longitudinally obtained clinical, serologic, and immunogenetic data on children with MCTD who were examined at our institution during a 20-year period, and we
correlate these findings with disease outcomes.
Selection of patients. Records of all children seen in
the Division of Immunology and Rheumatology at the University of Missouri Hospital and Clinics between 1969 and
1990 were reviewed by a physician. Patients selected for
analysis were those who were under the age of 18 years at
presentation to the Division of Immunology and Rheumatology, were found to have high-titer anti-RNP antibodies, and
had overlapping clinical features of juvenile rheumatoid
arthritis, systemic lupus erythematosus (SLE), dermatomyositis, or scleroderma, according to the criteria of Porter et a1
(4). Eleven patients who met these 3 criteria were identified
for further study.
Enzyme-linked immunosorbent assay (ELISA)and
immunoblotting for antinuclear antibodies. Sera were characterized for the presence of autoantibodies reactive with
Table 1. Clinical manifestations in the 11 children with mixed
connective tissue disease*
Swollen hands
Raynaud’s phenomenon
Abnormal carbon monoxide
diffusing capacity
Sclerodactyl y
Abnormal esophageal motilityt
Proximal muscle weakness
Pleural effusion
Cardiac disease
Malar erythema
Renal disease
Rheumatoid nodules
Central nervous system disease
At disease
over the
followup period
11 (100)
6 (55)
9 (82)
3 (27)
11 (100)
9 (82)
9 (82)
9 (82)
4 (36)
3 (27)
5 (45)
4 (36)
4 (36)
3 (27)
4 (36)
3 (27)
3 (27)
2 (18)
0 (0)
2 (18)
2 (18)
7 (64)
6 (55)
6 (55)
6 (55)
5 (45)
4 (36)
4 (36)
4 (36)
4 (36)
3 (27)
3 (27)f
2 (18)
2 (18)
* Values are the number (%) with the finding.
t Determined by barium esophagraphy, manometry, or both.
f Demonstrated by renal biopsy to be focal proliferative glomerulonephritis in 1 patient, membranous glomerulonephritis in 1 patient,
and diffuse proliferative glomerulonephritis in 1 patient.
U1-70-kd, A, B’/B, and D polypeptides using an snRNP
polypeptide-specific ELISA (2), and were analyzed by immunoblotting for reactivity with U1-70-kd, A, C, B‘/B, and
D polypeptides (5). Sera were also tested by double immunodiffusion for autoantibodies to SS-AIRo, SS-B/La, RNP,
Sm, PM-1 (PM-Scl), and Scl-70, by passive hemagglutination
for autoantibodies to extractable nuclear antigen, and by the
Crithidia luciliae method for autoantibodies to doublestranded DNA (dsDNA), as described (6).
Data collection and analysis. Clinical data were collected and stored using a standardized clinical database, as
previously described (6). Disease onset, course, and outcomes were categorized according to the character of the
clinical disease: mild (disease with no significant major organ
involvement or functional impairment), moderate (disease
with major organ involvement or functional impairment or
disability requiring intervention for accomplishing activities
of daily living), or severe (disease with major organ involvement and functional impairment or disability requiring intervention for accomplishing activities of daily living, or lifethreatening disease). Remission was defined as the absence
of clinical evidence of active disease on laboratory or
physical examination.
Statistical analysis. Fisher’s exact test and odds ratios
were used to analyze the HLA data. The Bonferroni correction was applied for multiple comparisons (6).
HLA typing. HLA typing was performed in 9 of the
patients, using a microcytotoxicity assay as previously described (6). Cells obtained from 102 healthy local Caucasian
blood donors were used as controls. HLA genotypes were
determined using HLA-DR2 and HLA-DR4 group-specific
primers in the polymerase chain reaction, as described
earlier (3).
Demographic characteristics of the patients.
Eleven patients (1 boy, 10 girls; all Caucasian) met the
criteria for inclusion in this study. The mean age at
symptom onset was 12.9 years (range 5-18 years), and
the mean duration of symptoms was 9.6 years (range
4-17 years). The mean length of time from the first
symptoms to diagnosis was 10.3 months (range 3-26
months), and the mean length of followup was 9.8
years (range 4-20 years).
Clinical and laboratory features. Clinical features at disease onset and cumulatively over the
followup period are summarized in Table 1. The
clinical manifestations found to be the most prevalent
were arthralgidarthritis, swollen hands, Raynaud’s
phenomenon, abnormal diffusing capacity for carbon
monoxide, and sclerodactyly. None of the patients
had diffuse sclerosis, although 1 had morphea. Laboratory findings at disease onset and cumulatively over
the followup period are summarized in Table 2. Results of ELISA testing for autoantibodies against the
(U)snRNP are shown in Figure 1.
Clinical outcomes. Following corticosteroid
treatment, 8 of the 11 patients had either mild disease
without significant functional impairment or were in
Table 2. Laboratory results in the 11 children with mixed connective tissue disease*
Anti-RNP autoantibodies
Anti-ENA autoantibodies
Anti-Sm autoantibodies
Anti-SS-A/Ro autoantibodies
Anti-SS-B/La autoantibodies
Anti-PM-1 autoantibodies
Anti-Scl-70 autoantibodies
Anti-dsDNA autoantibodies
Rheumatoid factor?
Anemia (Hgb <10 gm/dl)
Leukopenia (<4,500/mm3)
At disease
over the
followup period
11 (100)
I I (100)
0 (0)
2 (18)
0 (0)
0 (0)
11 (100)
11 (100)
1 (9)
3 (27)
0 (0)
0 (0)
0 (0)
0 (0)
3 (27)
2 (18)
8 (73)
1 (9)
1 (9)
1 (9)
1 (9)
8 (73)
7 (64)
3 (27)
4 (36)
2 (18)
* Values are the number (%) with the finding. Anti-ENA = antiextractable nuclear antigen; anti-dsDNA = anti-double-stranded
DNA; Hgb = hemoglobin.
t Titers were significantly elevated among all 8 patients.
f Complement levels were mildly depressed among all 7 patients.
70 kd
, g. d
.. .
Figure 1. Results of enzyme-linked immunosorbent assay (ELISA)
anti-Ul-70-kd, anti-A, anti-B'/B, and anti-D autoantibodies (IgG
and IgM) in the sera of 9 children with mixed connective tissue
disease. Values shown are the highest (in optical density units) for
each patient over the course of illness. Horizontal dotted lines
indicate the cutoff levels for local normal control sera. Serum from
1 patient was tested only for IgG autoantibodies. Sera obtained
during active disease were not available for ELISA in 2 patients;
both patients were previously shown by immunoblotting to have
anti-Ul-70-kd autoantibodies during active disease.
remission. Three of these 8 patients had a prolonged
remission, with a mean disease-free interval of 8.7
years (range 5-15 years); two of the 3 were taking no
medications, and 1 was taking 5 mg of prednisone
daily. One of the 11 patients had moderately active
disease, with disability due to arthritis, flexion contractures, and joint deformities. Two of the patients
died during the followup period. One died of pulmonary hypertension and pericarditis with cardiac tamponade, 10 years after MCTD was diagnosed. The
second, who had membranous glomerulonephropathy
and was receiving long-term immunosuppressive treat-
ment, died of a Pneurnocystis infection, 12 years after
diagnosis of MCTD.
Immunogenetics. Immunogenetic studies demonstrated that 6 of the 9 patients tested (67%) were
HLA-DR2 positive and 4 of 9 (44%) were HLA-DR4
positive; all 9 had either HLA-DR2 or HLA-DR4,
versus 53 of 102 normal controls ( P = 0.004, P,,, =
0.04, odds ratio 1.9). One patient had both DR2 and
In recent years, there have been significant
advances in the characterization of autoantibodies to
snRNPs, yielding data that were not available at the
time of earlier studies on childhood MCTD. In the
present study, an ELISA specific for individual snRNP polypeptides and immunoblotting were used to
characterize autoantibodies. The U1 RNP 70-kd
polypeptide antigen with which autoantibodies reacted
in this study has previously been shown to be distinct
from the 70-kd antigen with which some Sm-positive
SLE patient sera react. Using ELISA and immunoblotting on sera collected serially over a 20-year period
(Table 2 and Figure l), 11 of 11 patients had autoantibodies against the U1-70-kd polypeptide, while 3 of 11
(27%) had autoantibodies against the D polypeptide of
the Sm antigen, although only 1 showed anti-Sm
reactivity in the standard double immunodiffusion
assay. Nine of 11 sera (82%) were reactive to the B'/B
polypeptides, but it was conjectured (though not
proven) that in most, this reactivity represented specificity for an epitope distinct from that associated with
anti-Sm autoantibodies characteristic of SLE (5).
We have recently reported that among adult
CTD patients with high titers of autoantibodies against
the U1-70-kd antigen, as opposed to CTD patients
without these autoantibodies, there were significant
associations with the presence of sclerodactyly , swollen hands, Raynaud's phenomenon, telangiectasias,
and abnormal esophageal motility (6). In this regard,
all of the patients in the present study had autoantibodies against U1-70-kd and clinical features similar
to those in the adult patients with this autoantibody
(Table 1). De Rooij et a1 have recently studied 5
children with autoantibodies against U1-70-kd and
described findings similar to those reported here. All
of their patients had arthralgia or arthritis, swollen
hands, Raynaud's phenomenon, and pulmonary function abnormalities (7). Thus, it appears that, both for
children and for adults, there are similar clinical man-
ifestations among individuals with high titers of autoantibodies against the U1-70-kd polypeptide.
Nephritis, cardiac disease, thrombocytopenia,
diffuse sclerosis, and central nervous system disease
were uncommon or absent among the patients in the
present study, Renal disease (focal proliferative, diffuse proliferative, and membranous glomerulonephritis) occurred in only 3 of 11 patients and was confined
to those who had IgG autoantibodies against the D
polypeptide of snRNP and had HLA-DR2. Patients
with severe disease andlor fatal outcomes were also
those who developed autoantibodies against dsDNA
and the D polypeptide. Thus, the combination of
HLA-DR2, autoantibodies against the D polypeptide,
and autoantibodies against dsDNA appeared to identify patients in whom clinical renal disease would
develop and who would experienced adverse outcomes.
Among adult CTD patients, an association of
the presence of anti-U 1-70-kd autoantibodies and
select genotypes of HLA-DR4 and HLA-DR2 has
also been identified (3,6). Several previous studies
from the US and Europe have demonstrated a relationship between the presence of autoantibodies to
RNP and HLA-DR4 or HLA-DR2 (3,6,7-11). In the
present study, 100% of the 9 patients who were HLA
typed were positive for either HLA-DR2 or HLADR4, versus 53 of 102 controls. Despite the high
prevalence of arthritis (11 of 11 patients) and rheumatoid factor positivity (8 of 11 patients), there was a
relatively low frequency of HLA-DR4 in these patients (4 of 9). Furthermore, none had erosive arthritis,
similar to our findings among adult anti-U1-70-kd
autoantibody positive CTD patients (6).
In summary, our series of anti-U1-70-kd autoantibody positive pediatric MCTD patients showed a
high prevalence of arthralgidarthritis , swollen hands,
Raynaud’s phenomenon, abnormal pulmonary function, and sclerodactyly. In comparison with earlier
reports of less well-defined MCTD patient groups, the
overall severity of illness was mild to moderate and
clinical outcomes appeared favorable in the majority
of these children. The presence of autoantibodies to
dsDNA and IgG anti-D polypeptide autoantibodies
may, however, identify a subgroup of patients who are
more prone to develop renal disease and to have a
worse clinical outcome. The finding of an association
of HLA-DR4 and HLA-DR2 with disease susceptibility among these children is similar to that which has
been reported among adult patients with anti-U 170-kd autoantibodies and CTD (3,6).
We gratefully acknowledge the excellent technical
assistance of Don L. Hill.
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connection, clinical, serological, analysis, disease, children, longitudinal, tissue, mixed, autoantibodypositive, э70
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