TwoNP R 1 SistersExhibit Producing Anti-U Serological Concordance and Clinical Discordance MORRIS REICHLIN, PATRICIA ABUMOHOR and YASUHIKO ITOH Arthritis/Immunology Program, Department of Medicine, Oklahoma University Health
Sciences Center, Oklahoma Medical Research Foundation, 825 Northeast 13th Street, Oklahoma City, Oklahoma 73104, USA
Two sisters had autoimmune responses to the U RNP particle that were quantitatively similar 1 and/or identical in molecular characteristics. No other autoantibodies were demonstrable. Both sisters immunoprecipitated only U RNA, had a reaction of identity in gel diffusion, 1 bound the 68-kDa band in HeLa cell extract in Western blot, and reacted almost equally to a rabbit anti-idiotypic reagent made against either sister’s isolated anti-U RNP Fab frag1 ments. They both carried a DR 4 allele, which has been associated with anti-U RNP produc1 tion in several studies. While the sisters both had Raynaud’s phenomenon, their clinical pictures were otherwise dissimilar. One had a seizure disorder (Ju); the other had polymyositis and features of scleroderma (Je). In sister Ju, Raynaud’s phenomenon was manifest for the first time in association with the appearance of precipitating anti-U RNP. 1
Key Words: Sisters Anti-U RNP Autoimmunity Polymyositis SLE Overlap syndromes 1
Introduction
serologic hallmark of systemic lupus erythematosus (SLE) and its variants is the production of a variety of autoantibodies, some of which are associated with clinical subsets~. Among these, antibodies to the U, nuclear ribonudeoprotein occur in 40% of SLE patients and ~3-~t~~~o of patients with overlap features of SLE, polymyositis and scleroderma, designated mixed connective tissue disease O4CTD~&dquo;). Whatever the clinical picture, patients with predominantly anti- U 1 RNP have a low frequency of nephritis and a close association with Raynaud’s phenomenon 4. We have had the opportunity to study a family in which two sisters exhibited predominant autoimmune responses to the U,RNP particle but had very different clinical pictures, although both manifested Raynaud’s phenomenon. Detailed serological studies of the sisters and the asymptomatic
female with a 2-year history of classical Raynaud’s phewith three-phase color changes of her hands and lips on exposure to cold. She had also had swollen hands and stiffness in the small joints of her hands, elbows, shoulders and knees. She had lost ll lbs in weight and trouble up because of heavy legs and shortness of breath for 1 month. She also had trouble lifting things from high places and difficulty opening her mouth completely. There was no history of photosensitivity, alopecia, pleuritic chest pain, nausea and vomiting, seizures, burning on urination, hematuria, nomenon
The
parents reveal the presence of a very similar autoimmune response in the sisters and high titers of diverse autoantibodies in the parents. The concomitant appearance of
Raynaud’s phenomenon and high titers of anti-UtRNP one of the sisters suggests a possible immunopathogenetic role for this autoantibody in the evolution of this vascular abnormality. in
Case
description
Sister Je
.
The patient presented in July 1984 as a 20-year-old white Correspondence: Morris Reichlin, M.D., Oklahoma Medical Research Foundation, 825 Northeast 13th Street, Oklahoma City, Oklahoma 73104, USA.
any sensory changes. Her past medical history was notable only for a tonsillectomy at age 15. Her family history was that her father, age 41, and her mother, age 40, were healthy; her sister, who was then 16, had been healthy up to that time. She denied any allergies and did not use tobacco, alcohol or any other drugs. Her physical examination was notable for a blood pressure of 98/70, a pulse of 64 and a normal temperature. Her oral aperture was maximally 4 cm. She had normal hair, and fundi and there were no oral or nasal mucosal ulcers. Her thyroid was not enlarged, there was no lymphadenopathy and her chest was clear to and auscultation. Her breasts were symmetrical and without masses, and her heart examination revealed normal heart sounds and regular rhythm, and no rubs or murmurs. Her abdomen was soft and there was no liver or spleen enlargement. She had obvious cyanosis of her hands and feet and they were cold. She had puffy edema of all digits, and tender but not swollen metacarpophalangeal joints. The rest of her joint examination was normal except for general tenderness of the metatarsophalangeal joints. or
249
Downloaded from lup.sagepub.com at NORTH CAROLINA STATE UNIV on March 18, 2015
Her strength examination showed profound proximal weakness. Her neck flexors, deltoids, biceps, triceps and quadriceps were all 3/5. Her peripheral strength was also decreased, with grip strength of 94 mmHg on the right and 100 mmHg on the left as measured with a
sphygmomanometer. Her neurological examination was completely normal. Her serology is presented in Table I but she had an nRNP precipitin at the outset. Her laboratory studies revealed a normal urinalysis and complete blood count (CBC).
protein electrophoresis was notable for a polyclonal increase in gamma-globulin of 2.5 g 0/0. Her creatine phosphokinase (CPK) was 2050 U/1 (normal 0-120), LDH 481 (normal 60-200), SCOT (AST) 121 (normal 0-30) and SGPT (ALT) 132 (normal 4-40). Her Her
serum
electromyogram showed normal nerve conduction but increased irritability with a small amount of partial denervation potentials. There were motor unit potentials of brief duration and small amplitude polymorphic configuration. The muscle biopsy showed muscle fiber necrosis with a mononucleolar infiltrate both within and around fibers as well as around blood vessels. There were also regenerating fibers. The diagnosis of polymyositis was made and she was placed on prednisone (50 mg q.d.) in July 1984. By 11 October 1984 her strength and CPK had returned to normal and the prednisone was switched to a dose of 50 mg every other day. Over a period of 1 year the medication was tapered and discontinued in October 1985. The patient was asymptomatic until January 1991 when she had the re-emergence of profound weakness without a CPK elevation. She was treated again with daily prednisone and had a dramatic response within 2 months; she is presently on 30 mg prednisone q.o.d. and is doing well.
Sister Ju
Sister Ju was first seen as a patient in May 1989 after the development of Raynaud’s phenomenon in the preceding 6 months. Her medical history began in February 1986 when she developed grand mal seizures, with subsequent episodes in February and August 1988. They were characterized by loss of consciousness, tonic clonic movements, tongue biting, urinary incontinence, post ictal headache and confusion. Computerized tomography with contrast of the head and an eldctroencephalogram (EEG) were both normal. Later a sleep-deprived EEG was done, which was also normal. When 21 years old, the patient was on dilantin for the first 18 months and since March 1988 she has been on divalproex (500 mg/ day). She developed typical Raynaud’s phenomenon in November 1988 and has had worsening symptoms since. She had a motorcycle accident when she was 13 years old, suffering a blow to the left temple, but did not lose consciousness. In the same year she was in an automobile accident and hit her head but had no lacerations and did not lose consciousness. She began having ’out of body’ experiences about 7 years ago. These were at first weekly but more recently occur only once every 3 or 4 months. There is no loss of consciousness, abnormal movements, tongue biting or urinary incontinence. She has been treated for peptic ulcer disease since the age of 13. She has had few symptoms recently and occasionally takes citruc~i. She had asthma as a child, but has had no problems with asthma since the age of 13. She uses no tobacco or alcohol and is allergic to penicillin. She denies photosensitivity, sicca, bleeding anywhere (except for normal menses), arthritis, skin rash, pleuritic chest pain, __
dyspnea Table I
or
genito-urinary problems.
She had a tonsillectomy as a child. She had a leftknee arthroscopy with a lateral release with good results. She was a ballet major in college. She still dances
Serological data of sister Je.
actively. Her physical examination was normal except for some mild facial erythema and a small amount of fluid on the left knee. There was no evidence of Raynaud’s phenomenon at that time, but her history was classical for the three-phase color changes, and she subsequently exhibited typical pallor, cyanosis and rubor with or without cold provocation. Her initial and subsequent urinalysis, CBC and Chem 18 were all normal. Her serological findings are presented in Table II but she developed an nRNP precipitin in May 1989 that she had not had in August 1986 and November 1987 when tested as part of an ongoing family study in our clinic. She has been treated with nifedipine (10 mg t.i.d.) for her Raynaud’s phenomenon, which was moderately effective. Her facial erythema was never again apparent and she’ continues on divalproex for her
ANA: ~.ntinucle~r antibodies; Ppt. Ab: precipitating antibody; HEp-2: human epithelial-2 cell line; Neg: negative.
250
Downloaded from lup.sagepub.com at NORTH CAROLINA STATE UNIV on March 18, 2015
Table ~I
Serological data of sister Ju.
Abbreviations
are
explained
in the footnote to Table I.
bled from the central ear artery on days 6, 7 and 8 after the iv injection. The sera were then absorbed by passage over a column of IgG-sepharose in which the IgG was Cohn fraction until the sera no longer reacted with normal IgG or normal Fab2 in enzymelinked immunosorbent assay (ELISA).
seizure disorder. She was seen by a neurologist in April 1990 for a follow-up of her seizure disorder, although she has had no seizures since 1988. Because of a history of fatigue precipitating her seizures, the neurologist thought the diagnosis might be juvenile myoclonic epilepsy. She has developed no other features characteristic of SLE. HLA typing was performed by the microcytotoxicity method of Terasaki with commercial plates for the determination of HLA-A, B, C and D.
were
Serological
Preparation of ~z~a~i-~T~lml~F~ autoantibodies
Specific anti-UtRNP autoantibodies were prepared as the ~’a~2 fragments by the following method. IgG (100mg) from a monospecific anti-U, ~.N~’ serum was coupled to cyanogen bromide activated sepharose (10 g) according to the manufacturer’s instructions. The column was charged with calf thymus extract ~1~3 ~~~ and washed extensively until.the optical density (OD) of the effluent was less than 0.01 at 280 nm. Serum from sister Je (10 cc) was applied to the column and washed extensively. The ~~ ~&dquo;-aa~ta-~.T,~~~&dquo; complexes were then eluted with ~. ~ M acetic acid. The eluted complexes were adjusted to pH 4.5 with 6 N I~Ia~i~ added dropwise. The complexes were digested with pepsin ~~~1~ of pepsin/wt complexes) for 18 h at 3~°C. The pepsin was then inactivated by dialysis against 1 (~f~ volumes of 0. 1 M
buffer, 8.0. The same procedure wax used to prepare purified anfi-UIRNP tibodies from sister Ju as well as other patients with anti-U,RNP. Preparation c~,~’ c~ra~i-i~i~~,~~i~ (anti-URNP) serum Rabbits were immunized with 0.2 mg anti-UtRNP Fab,, in 1 cc of phosphate buffered saline (PBS) and an equal volume of complete Freud’s adjuvant. The emulsion was injected into multiple intramuscular and subcutaneous sites. At 3 weeks a second injection of 0.2 mg ~&dquo;a~~ in 1 cc PBS was iu~~~t~d iutra~~uuu~I~ (iv). The rabbits
tests
Indirect immunofluorescence on mouse kidney, HEp-2 cells and Crithidia luciliae and precipitating antibodies were measured by previously described methods’. Antibodies to single-stranded DNA (ssDNA) were measured with a modified assay6. Western blotting and RNA
immunoprecipitation
were
performed by published
methods’.
Results
Serological findings The serological profiles on the two sisters are presented in Tables I and n. Sister Je, who presented with polymyositis, sclerodactyly and Raynaud’s phenomenon, had a high-titer nuclear speckled antinuclear antibody (ANA) test at the outset, negative tests for anti-DNA, and a single precipitin line identified as anti-nRNP. Her serological findings have been relatively constant throughout the course of her disease. The second sister Ju was first seen as part of a family study. On 7 August 1986 and 23 November 1987 her routine findwere normal but on 3 May 1989 her ANA test on mouse kidney, which had been previously negative, was found to be 1/4860 and was 1/9720 on cells, both with a nuclear speckled pattern. On that and subsequent occasions, her has had an nRNP precipitin that formed a reaction of identity with her sister’s serum and a prototype immunospecific anti-nRNP (anti-UIRNP) serum. Thereafter her serological profile remained constant and was identical quantitatively and almost qualitatively to her sister’s serological findings. Although
251
Downloaded from lup.sagepub.com at NORTH CAROLINA STATE UNIV on March 18, 2015
neither parent had anti-nRNP precipitins, they both had positive ANA tests, which exhibited immunofluorescent patterns different from each other and from their daughters’. The mother had a positive ANA test on HEp-2 cells with a titer ranging from 1/360 to 1/1080 with a nuclear fine speckled pattern, while the father had a positive ANA test on HEp-2 cells with a titer of 1/4860 and a nucleolar pattern. Neither had a positive precipitin test and neither had antibodies to either ssDNA or double-stranded DNA.
.~4 rcti- ~~~l~t.F’‘ ELISA strongly positive ELISA for antibodies samples. The reactivity of her serum UIRNP was inhibited by more than 90% by affinity-purified bovine nRNP when a dilution of her serum (10 -4) that would yield an OD of 1 on U¡RNP control plates was mixed with 10 ¡.Lg/ml of affinity-purified bovine UIRNP. Interestingly, the serum of sister Ju had an elevated level of anti-U,RNP by ELISA in the serum samples of 19 August 1986 and 19 November 1987. This activity was blocked by more than 9!~~1~ by the addition of 10 ¡Lg/ml affinity-purified bovine U1RNP and was thus considered specific anti-U1RNP. Between 19 November 1987 and 19 May 1989, the ELISA titer of anti-U1RNP rose 20-fold in association with the development of Raynaud’s phenomenon. These data are sumSister Je had
to
on
a
1 Silver stain of a 7 M urea 10% po!yacry!amide gel with RNA extracted from immunoprecipitates made from HeLa extract and family members’ sera. Lane l: total HeLa cell RNA; lane 2: antiU I RNP serum; lane 3: anti-Ro/SSA serum; lane 4: sera of sister Je in 1987; lane 5: sera of sister Je in 1989; lane 6: sera of sister Ju in 19~~; lane 7: sera of sister Ju in 1989; lane 8: father; lane 9: mother; lane 10: normal human serum. Note that sister Ju was very weakly positive for anti-UiT~NA in 1986 but strongly positive in 1989. Sister Je’s sera were all strongly positive for anti-UIRNA.
Figure
all
and 9 are the results with the parents’ sera, and lane 10 is a normal human serum. All sera of sister Ju subsequent to 19 May 1989 immunoprecipitate U,RNA. Western blot
In Figure 2 are seen the reactions of the family members’ in Western blot with Hela cell extract as antigen. In lanes 13-15 are three different samples of Je’s sera, which show an intense band at 68 kDa and a weak band at 75 kDa. Sister Ju’s sera in lanes 10-12 stain a protein aaf ~-~- 65 kDa in the first two samples when her antiUII~.I~P titer was elevated but not yet capable of precipitating U,RNP. In the 19 May 1989 sample, staining of the 68-kDa- band (lane 12) is seen for the first time. Her sera also stain 100-kDa and 52-kDa bands weakly. The father’s serum in lanes 7-9 stain bands of 85 and 48 kDa, while the mother’s sera in lanes 5 and 6 intensely stain a 75-kDa band. The molecular natures of none of the bands, except for the 68-kDa band, are known. Interestingly, our prototype anti-nRNP serum has a band that migrated slightly slower (and is therefore presumably larger) than the 75-kDa band seen in the mother’s serum and that of Je.
marized in Table III. The parents had borderline values of anti-U,RNP but in neither case was the activity significantly inhibited by affinity-purified U,RNP. RNA The
sera
immunoprecipitation
sera
of the family members
were
subjected to RNA
immunoprecipitation and the results are seen in Figure 1. Sister Ju’s serum in all samples precipitated UIRNA, and two representative samples are seen in lanes 6 (9 August 1986) and 7 (19 May 1989). The first sample of Sister Je (lane 4) may immunoprecipitate a faint U,I~~~ band but the 19 May 1989 sample (lane 5) in3rnun~~ire~ipitat~s an intense U ¡RNA band. Lanes 8 m ELISA values for members’ sera.
anti-U1RNP
in
analysis
family
Idiotypic studies of the sisters’ ~r~~i-~I,.~.l~~’ and Fab fragments Rabbit serum 226 was prepared by immunization with Ju anti-UIRNP Fab. When absorbed with Cohn fil IgG, this did not react significantly with normal Fab2. as seen in Table III. In all cases, 5 Ag Fab fragments/ml were used to coat the plates and then the absorbed rabbit
Upper limit of normal values is 104 Ulml. Precipitation usually occurs with sera having at least 2 x 105 ~7‘~mt. Sister Ju’s anti-U¡RNP titer rose between November 1987 and May 1989 in association with development of Raynaud’s phenomenon.
252
Downloaded from lup.sagepub.com at NORTH CAROLINA STATE UNIV on March 18, 2015
Table IV Comparison of various anti-U,RNP coats in reaction with anti-idiotype (Id) reagent No. 226 in ELISA.
Anti-UIRNP antibodies
react strongly with anti-M reagent; antiRo/SSA and anti-La/SSB antibodies react poorly with angi-I~ reagents. Values listed are OD at 405 nm in the anti-Id ELISA.
Table V
HLA
profiles
on
family.
Figure 2 Western blot analysis with HeLa cell
extract and family members’ sera. Lane 1: normal human serum; lanes 2+3: antiRo/SSA and anti-La/SSB sera; lane 4: anti-nRNP serum; lane 5: mother’s sera in 1986; lane 6: mother’s sera in 1989; lane 7: father’s sera in 1986; lane 8: father’s sera in 1987; lane 9: father’s sera in 1989; lane 10: sister Ju’s sera in 1986; lane I1: sister Ju’s sera in 1987; lane 12: sister Ju’s sera in 1989; lane 13: sister Je’s sera in 1986; lane 14: sister Je’s sera in 1987; lane 15: sister Je’s sera in 1989. Sister Je has anti-68 kDa in all sera. Sister Ju has anti-65 kDa in all sera but makes anti-68 kDa in 1989 sample. Mother has antibody to a 75-kDa protein in HeLa
been previously associated with the several studies 8-11.
extract.
D R4DQw3
alleles in
o
Discussion
added at dilutions of 10-2 and 10 -’. After washing, goat anti-rabbit IgG alkaline phosphatase was added. After appropriate incubation and washing, paranitrophenyl-phosphate (substrate) was added and the OD values at 405 nm read at 1 h with a Multitek serum was
are several points that make the study of this family notable. Much has been made c~:~ the relationship
There
of antibodies to U,RNP and its 68-kDa polypeptide and the syndrome of MCTD. Indeed, sister Je had polymyositis and features of scleroderma (sclerodactyly, small oral aperture and Raynaud’s phenomenon). The second sister, however, did not satisfy criteria for any disease but exhibited a grand mal seizure disorder and Raynaud’s phenomenon. Her clinical picture has been stable; her seizure disorder is well controlled with anticonvulsant medications and her Raynaud’s phenomenon has been treated with procardia with moderate success. Thus, their very similar autoimmune are associated with quite different clinical pictures, except that they both manifest ~.a~naud’~ phenomenon. Such data that, except for Raynaud’s phenomenon, there is no mechanistic relationship between clinical expression of disease and antibodies to ~.1~~.I~I~’. Finally, the presence of substantial titers of unrelated autoantibodies in the two parents raises anew the question of the significance of such specificities in asymptomatic first-degree relatives. An ELISA study probing the relationship of genetic factors in the major histocompatibility complex and specific autoantibodies in asymptomatic first-degree, relatives in SLE and Sj6gren’s
scanner.
As seen, all anti-UtRNP Fabs tested were reactive, but the sisters’ UiRNP Fab fragments were more antigenic than those from other patients. Anti-Ro/SSA and anti-La/SSB Fab fragments were non-reactive. Each sister was slightly superior ~~t~~~~i~~lly to the other sister when the serum prepared from their individual anti-U,.RNP Fab fragments were used to react with plates coated with their c~~~ ~,~.~~ Fab. This suggests that there are idiotypic determinants characteristic and cross-reactive of the anti-UIRNP specificity but also individually specific determinants by the two
sisters. HLA testing The results of HLA testing of the lymphocytes of the family members are listed in Table IV. As shown, the sisters were non-identical but shared the ~i~ss I alleles ~~~~~-, and the class 11 alleles DR.DQw3’ which they received from the mother. Antibodies to ~J,i~~fi~ have
253
Downloaded from lup.sagepub.com at NORTH CAROLINA STATE UNIV on March 18, 2015
syndrome families showed that ~5~0 of these relatives were anti-Ro/SSA positive and 90% had either DR2 or DR3 alleles 12. Thus, the same HLA-D alleles associated with high levels of anti-Ro/SSA in patients were associated with low but elevated levels of anti-Ro/SSA in first-degree relatives. Whether this confers a greater risk for the development of disease in these relatives remains to be seen. At least it can be concluded that autoantibody production in first-degree relatives has, in part, a genetic basis. We have not found the presence of antinucleolar antibodies in the parents of other patients who produce anti-U,RNP (or for that matter other autoantibodies such as anti-Sm, anti-Ro/SSA etc. in the lupus spectrum). In our families, about 25% of firstdegree asymptomatic relatives have a positive ANA test but none thus far with a nucleolar pattern. We have, however, found some other first-degree relatives as well as some SLE patients who also produce antibodies to a 75-kDa protein as did the mother and sister Je. Studies are underway to identify the molecular nature of this antigenic target to see what insights this information could reveal about the mechanism of autoimmune disease. The most striking finding in this family was the development of Raynaud’s phenomenon in sister Ju coincident with a 20-fold increase in the titer of anti-U IRNP. While recent studies of patient populations have shown a strong association of Raynaud’s phenomenon and anti-U1RNp2-4, this is the first reported example of a temporal relationship of a large increase of anti-UIRNP and the de novo development of Raynaud’s phenomenon. This suggests that anti-U1RNP itself or an associated antibody or other associated biological mediator is involved in the pathogenesis of Raynaud’s
most important lesson from this family is that there is much to be learned from long-term study studies that permit the observation of the transifamily tions from asymptomatic relative to patient that will occur in families with autoimmune diseases. The study of such families should improve our understanding of the relationship between genes and autoantibody production on the one hand and between autoantibodies and clinical expression on the other.
Perhaps the
Acknowledgements This work was supported by NIH grants POI A-21568 and ROt .P~~-31133.
References 1. Reichlin M.
2.
3.
4.
5.
6.
7.
8.
phenomenon. It is also quite interesting that, while the HLA profile of the two sisters is not identical, the autoimmune response was quite similar. The titers in both ELISA and ANA tests reflecting the antibody to U1RNP were quite similar, as were the RNAs immunoprecipitated and the proteins bound in Western blot. Even the idiotypic fine specificity of their anti-U,~I~T~ molecules was closely related, similar and distinctive amino acid sequences in the V region of either of their heavy and light chains or both. Since the two sisters shared the haplotype A2B27C2DR4DQW3, it appears that the DR, and/or the DQW3 play a crucial role in determining the precise molecular character of the autoimmune response.
9.
10.
11.
12.
Systemic lupus erythematosus.
In:
Bigazzi PE,
Reichlin M, eds. Systemic A utoimmunity. New York, Basel, Hong Kong: Marcel Dekker Inc., 1991: 163-99. Habets JW, De Rooij DJ, Salden MH et al. Antibodies against distinct nuclear matrix proteins are characteristic for mixed connective tissue disease. Clin Exp Immunol 1983 ; 54: 265-76. Petterson I, Wang G, Smith EI et al. The use of immunoblotting and immunoprecipitation of (U) small nuclear ribonucleoproteins with mixed connective tissue disease and systemic lupus erythematosus. Arthritis Rheum 1986; 29: 986-96. Reichlin M, Van Venrooij WJ. Autoantibodies to the URNP particles: relationship to clinical diagnosis and nephritis. Clin Exp 1991; 83: 286-90. Immunol Maddison PJ, Provost TT, Reichlin M. Serological findings in patients with ’ANA-negative’ systemic lupus erythematosus. Medicine 1981; 60: 87-94. Kulick K, Reichlin M. Antibodies to single-stranded DNA in patients with discoid lupus erythematosus. Arthritis Rheum 1982; 25: 639-46. Itoh Y, Kriet JD, Reichlin M. Organ distribution of the Ro/SSA antigen in the guinea pig. Arthritis Rheum 1990; 33: 1815-21. Nishikai M, Sekiguchie S. Relationship of autoantibody expression and HLA phenotype in Japanese patients with connective tissue disease. Arthritis Rheum 1985; 28: 579-81. Smolen JS, Klippel JA, Penner E et al. HLA DR antigens in systemic lupus erythematosus: association with specificity of autoantibody responses to nuclear antigens. Ann Rheum Dis 1987; 46: 457-62. Harley JB, Sestak AL, Willis LG, Fu SM, Hausen JA, Reichlin M. A model for disease heterogeneity in systemic lupus erythematosus. Arthritis Rheum 1989; 32: 826-36. Olsen ML, Arnett FC, Reveille JD. Anti Sm and RNP antibodies are associated with distinct and different DQa and DQβ genes. Arthritis Rheum 1990; 33: 100 (abstract B169). Arnett FC, Hamilton RG, Reveille JD, Bias WB, Harley JB, Reichlin M. Genetic studies of Ro(SS-A) and La(SS-B) autoantibodies in families with systemic lupus erythematosus and
primary Sjögren’s syndrome.
Arthritis Rheum 1989; 32: 413-9.
(Received 3 March 1992) (Accepted 17 April 1992)
254
Downloaded from lup.sagepub.com at NORTH CAROLINA STATE UNIV on March 18, 2015
Sciences Center, Oklahoma Medical Research Foundation, 825 Northeast 13th Street, Oklahoma City, Oklahoma 73104, USA
Two sisters had autoimmune responses to the U RNP particle that were quantitatively similar 1 and/or identical in molecular characteristics. No other autoantibodies were demonstrable. Both sisters immunoprecipitated only U RNA, had a reaction of identity in gel diffusion, 1 bound the 68-kDa band in HeLa cell extract in Western blot, and reacted almost equally to a rabbit anti-idiotypic reagent made against either sister’s isolated anti-U RNP Fab frag1 ments. They both carried a DR 4 allele, which has been associated with anti-U RNP produc1 tion in several studies. While the sisters both had Raynaud’s phenomenon, their clinical pictures were otherwise dissimilar. One had a seizure disorder (Ju); the other had polymyositis and features of scleroderma (Je). In sister Ju, Raynaud’s phenomenon was manifest for the first time in association with the appearance of precipitating anti-U RNP. 1
Key Words: Sisters Anti-U RNP Autoimmunity Polymyositis SLE Overlap syndromes 1
Introduction
serologic hallmark of systemic lupus erythematosus (SLE) and its variants is the production of a variety of autoantibodies, some of which are associated with clinical subsets~. Among these, antibodies to the U, nuclear ribonudeoprotein occur in 40% of SLE patients and ~3-~t~~~o of patients with overlap features of SLE, polymyositis and scleroderma, designated mixed connective tissue disease O4CTD~&dquo;). Whatever the clinical picture, patients with predominantly anti- U 1 RNP have a low frequency of nephritis and a close association with Raynaud’s phenomenon 4. We have had the opportunity to study a family in which two sisters exhibited predominant autoimmune responses to the U,RNP particle but had very different clinical pictures, although both manifested Raynaud’s phenomenon. Detailed serological studies of the sisters and the asymptomatic
female with a 2-year history of classical Raynaud’s phewith three-phase color changes of her hands and lips on exposure to cold. She had also had swollen hands and stiffness in the small joints of her hands, elbows, shoulders and knees. She had lost ll lbs in weight and trouble up because of heavy legs and shortness of breath for 1 month. She also had trouble lifting things from high places and difficulty opening her mouth completely. There was no history of photosensitivity, alopecia, pleuritic chest pain, nausea and vomiting, seizures, burning on urination, hematuria, nomenon
The
parents reveal the presence of a very similar autoimmune response in the sisters and high titers of diverse autoantibodies in the parents. The concomitant appearance of
Raynaud’s phenomenon and high titers of anti-UtRNP one of the sisters suggests a possible immunopathogenetic role for this autoantibody in the evolution of this vascular abnormality. in
Case
description
Sister Je
.
The patient presented in July 1984 as a 20-year-old white Correspondence: Morris Reichlin, M.D., Oklahoma Medical Research Foundation, 825 Northeast 13th Street, Oklahoma City, Oklahoma 73104, USA.
any sensory changes. Her past medical history was notable only for a tonsillectomy at age 15. Her family history was that her father, age 41, and her mother, age 40, were healthy; her sister, who was then 16, had been healthy up to that time. She denied any allergies and did not use tobacco, alcohol or any other drugs. Her physical examination was notable for a blood pressure of 98/70, a pulse of 64 and a normal temperature. Her oral aperture was maximally 4 cm. She had normal hair, and fundi and there were no oral or nasal mucosal ulcers. Her thyroid was not enlarged, there was no lymphadenopathy and her chest was clear to and auscultation. Her breasts were symmetrical and without masses, and her heart examination revealed normal heart sounds and regular rhythm, and no rubs or murmurs. Her abdomen was soft and there was no liver or spleen enlargement. She had obvious cyanosis of her hands and feet and they were cold. She had puffy edema of all digits, and tender but not swollen metacarpophalangeal joints. The rest of her joint examination was normal except for general tenderness of the metatarsophalangeal joints. or
249
Downloaded from lup.sagepub.com at NORTH CAROLINA STATE UNIV on March 18, 2015
Her strength examination showed profound proximal weakness. Her neck flexors, deltoids, biceps, triceps and quadriceps were all 3/5. Her peripheral strength was also decreased, with grip strength of 94 mmHg on the right and 100 mmHg on the left as measured with a
sphygmomanometer. Her neurological examination was completely normal. Her serology is presented in Table I but she had an nRNP precipitin at the outset. Her laboratory studies revealed a normal urinalysis and complete blood count (CBC).
protein electrophoresis was notable for a polyclonal increase in gamma-globulin of 2.5 g 0/0. Her creatine phosphokinase (CPK) was 2050 U/1 (normal 0-120), LDH 481 (normal 60-200), SCOT (AST) 121 (normal 0-30) and SGPT (ALT) 132 (normal 4-40). Her Her
serum
electromyogram showed normal nerve conduction but increased irritability with a small amount of partial denervation potentials. There were motor unit potentials of brief duration and small amplitude polymorphic configuration. The muscle biopsy showed muscle fiber necrosis with a mononucleolar infiltrate both within and around fibers as well as around blood vessels. There were also regenerating fibers. The diagnosis of polymyositis was made and she was placed on prednisone (50 mg q.d.) in July 1984. By 11 October 1984 her strength and CPK had returned to normal and the prednisone was switched to a dose of 50 mg every other day. Over a period of 1 year the medication was tapered and discontinued in October 1985. The patient was asymptomatic until January 1991 when she had the re-emergence of profound weakness without a CPK elevation. She was treated again with daily prednisone and had a dramatic response within 2 months; she is presently on 30 mg prednisone q.o.d. and is doing well.
Sister Ju
Sister Ju was first seen as a patient in May 1989 after the development of Raynaud’s phenomenon in the preceding 6 months. Her medical history began in February 1986 when she developed grand mal seizures, with subsequent episodes in February and August 1988. They were characterized by loss of consciousness, tonic clonic movements, tongue biting, urinary incontinence, post ictal headache and confusion. Computerized tomography with contrast of the head and an eldctroencephalogram (EEG) were both normal. Later a sleep-deprived EEG was done, which was also normal. When 21 years old, the patient was on dilantin for the first 18 months and since March 1988 she has been on divalproex (500 mg/ day). She developed typical Raynaud’s phenomenon in November 1988 and has had worsening symptoms since. She had a motorcycle accident when she was 13 years old, suffering a blow to the left temple, but did not lose consciousness. In the same year she was in an automobile accident and hit her head but had no lacerations and did not lose consciousness. She began having ’out of body’ experiences about 7 years ago. These were at first weekly but more recently occur only once every 3 or 4 months. There is no loss of consciousness, abnormal movements, tongue biting or urinary incontinence. She has been treated for peptic ulcer disease since the age of 13. She has had few symptoms recently and occasionally takes citruc~i. She had asthma as a child, but has had no problems with asthma since the age of 13. She uses no tobacco or alcohol and is allergic to penicillin. She denies photosensitivity, sicca, bleeding anywhere (except for normal menses), arthritis, skin rash, pleuritic chest pain, __
dyspnea Table I
or
genito-urinary problems.
She had a tonsillectomy as a child. She had a leftknee arthroscopy with a lateral release with good results. She was a ballet major in college. She still dances
Serological data of sister Je.
actively. Her physical examination was normal except for some mild facial erythema and a small amount of fluid on the left knee. There was no evidence of Raynaud’s phenomenon at that time, but her history was classical for the three-phase color changes, and she subsequently exhibited typical pallor, cyanosis and rubor with or without cold provocation. Her initial and subsequent urinalysis, CBC and Chem 18 were all normal. Her serological findings are presented in Table II but she developed an nRNP precipitin in May 1989 that she had not had in August 1986 and November 1987 when tested as part of an ongoing family study in our clinic. She has been treated with nifedipine (10 mg t.i.d.) for her Raynaud’s phenomenon, which was moderately effective. Her facial erythema was never again apparent and she’ continues on divalproex for her
ANA: ~.ntinucle~r antibodies; Ppt. Ab: precipitating antibody; HEp-2: human epithelial-2 cell line; Neg: negative.
250
Downloaded from lup.sagepub.com at NORTH CAROLINA STATE UNIV on March 18, 2015
Table ~I
Serological data of sister Ju.
Abbreviations
are
explained
in the footnote to Table I.
bled from the central ear artery on days 6, 7 and 8 after the iv injection. The sera were then absorbed by passage over a column of IgG-sepharose in which the IgG was Cohn fraction until the sera no longer reacted with normal IgG or normal Fab2 in enzymelinked immunosorbent assay (ELISA).
seizure disorder. She was seen by a neurologist in April 1990 for a follow-up of her seizure disorder, although she has had no seizures since 1988. Because of a history of fatigue precipitating her seizures, the neurologist thought the diagnosis might be juvenile myoclonic epilepsy. She has developed no other features characteristic of SLE. HLA typing was performed by the microcytotoxicity method of Terasaki with commercial plates for the determination of HLA-A, B, C and D.
were
Serological
Preparation of ~z~a~i-~T~lml~F~ autoantibodies
Specific anti-UtRNP autoantibodies were prepared as the ~’a~2 fragments by the following method. IgG (100mg) from a monospecific anti-U, ~.N~’ serum was coupled to cyanogen bromide activated sepharose (10 g) according to the manufacturer’s instructions. The column was charged with calf thymus extract ~1~3 ~~~ and washed extensively until.the optical density (OD) of the effluent was less than 0.01 at 280 nm. Serum from sister Je (10 cc) was applied to the column and washed extensively. The ~~ ~&dquo;-aa~ta-~.T,~~~&dquo; complexes were then eluted with ~. ~ M acetic acid. The eluted complexes were adjusted to pH 4.5 with 6 N I~Ia~i~ added dropwise. The complexes were digested with pepsin ~~~1~ of pepsin/wt complexes) for 18 h at 3~°C. The pepsin was then inactivated by dialysis against 1 (~f~ volumes of 0. 1 M
buffer, 8.0. The same procedure wax used to prepare purified anfi-UIRNP tibodies from sister Ju as well as other patients with anti-U,RNP. Preparation c~,~’ c~ra~i-i~i~~,~~i~ (anti-URNP) serum Rabbits were immunized with 0.2 mg anti-UtRNP Fab,, in 1 cc of phosphate buffered saline (PBS) and an equal volume of complete Freud’s adjuvant. The emulsion was injected into multiple intramuscular and subcutaneous sites. At 3 weeks a second injection of 0.2 mg ~&dquo;a~~ in 1 cc PBS was iu~~~t~d iutra~~uuu~I~ (iv). The rabbits
tests
Indirect immunofluorescence on mouse kidney, HEp-2 cells and Crithidia luciliae and precipitating antibodies were measured by previously described methods’. Antibodies to single-stranded DNA (ssDNA) were measured with a modified assay6. Western blotting and RNA
immunoprecipitation
were
performed by published
methods’.
Results
Serological findings The serological profiles on the two sisters are presented in Tables I and n. Sister Je, who presented with polymyositis, sclerodactyly and Raynaud’s phenomenon, had a high-titer nuclear speckled antinuclear antibody (ANA) test at the outset, negative tests for anti-DNA, and a single precipitin line identified as anti-nRNP. Her serological findings have been relatively constant throughout the course of her disease. The second sister Ju was first seen as part of a family study. On 7 August 1986 and 23 November 1987 her routine findwere normal but on 3 May 1989 her ANA test on mouse kidney, which had been previously negative, was found to be 1/4860 and was 1/9720 on cells, both with a nuclear speckled pattern. On that and subsequent occasions, her has had an nRNP precipitin that formed a reaction of identity with her sister’s serum and a prototype immunospecific anti-nRNP (anti-UIRNP) serum. Thereafter her serological profile remained constant and was identical quantitatively and almost qualitatively to her sister’s serological findings. Although
251
Downloaded from lup.sagepub.com at NORTH CAROLINA STATE UNIV on March 18, 2015
neither parent had anti-nRNP precipitins, they both had positive ANA tests, which exhibited immunofluorescent patterns different from each other and from their daughters’. The mother had a positive ANA test on HEp-2 cells with a titer ranging from 1/360 to 1/1080 with a nuclear fine speckled pattern, while the father had a positive ANA test on HEp-2 cells with a titer of 1/4860 and a nucleolar pattern. Neither had a positive precipitin test and neither had antibodies to either ssDNA or double-stranded DNA.
.~4 rcti- ~~~l~t.F’‘ ELISA strongly positive ELISA for antibodies samples. The reactivity of her serum UIRNP was inhibited by more than 90% by affinity-purified bovine nRNP when a dilution of her serum (10 -4) that would yield an OD of 1 on U¡RNP control plates was mixed with 10 ¡.Lg/ml of affinity-purified bovine UIRNP. Interestingly, the serum of sister Ju had an elevated level of anti-U,RNP by ELISA in the serum samples of 19 August 1986 and 19 November 1987. This activity was blocked by more than 9!~~1~ by the addition of 10 ¡Lg/ml affinity-purified bovine U1RNP and was thus considered specific anti-U1RNP. Between 19 November 1987 and 19 May 1989, the ELISA titer of anti-U1RNP rose 20-fold in association with the development of Raynaud’s phenomenon. These data are sumSister Je had
to
on
a
1 Silver stain of a 7 M urea 10% po!yacry!amide gel with RNA extracted from immunoprecipitates made from HeLa extract and family members’ sera. Lane l: total HeLa cell RNA; lane 2: antiU I RNP serum; lane 3: anti-Ro/SSA serum; lane 4: sera of sister Je in 1987; lane 5: sera of sister Je in 1989; lane 6: sera of sister Ju in 19~~; lane 7: sera of sister Ju in 1989; lane 8: father; lane 9: mother; lane 10: normal human serum. Note that sister Ju was very weakly positive for anti-UiT~NA in 1986 but strongly positive in 1989. Sister Je’s sera were all strongly positive for anti-UIRNA.
Figure
all
and 9 are the results with the parents’ sera, and lane 10 is a normal human serum. All sera of sister Ju subsequent to 19 May 1989 immunoprecipitate U,RNA. Western blot
In Figure 2 are seen the reactions of the family members’ in Western blot with Hela cell extract as antigen. In lanes 13-15 are three different samples of Je’s sera, which show an intense band at 68 kDa and a weak band at 75 kDa. Sister Ju’s sera in lanes 10-12 stain a protein aaf ~-~- 65 kDa in the first two samples when her antiUII~.I~P titer was elevated but not yet capable of precipitating U,RNP. In the 19 May 1989 sample, staining of the 68-kDa- band (lane 12) is seen for the first time. Her sera also stain 100-kDa and 52-kDa bands weakly. The father’s serum in lanes 7-9 stain bands of 85 and 48 kDa, while the mother’s sera in lanes 5 and 6 intensely stain a 75-kDa band. The molecular natures of none of the bands, except for the 68-kDa band, are known. Interestingly, our prototype anti-nRNP serum has a band that migrated slightly slower (and is therefore presumably larger) than the 75-kDa band seen in the mother’s serum and that of Je.
marized in Table III. The parents had borderline values of anti-U,RNP but in neither case was the activity significantly inhibited by affinity-purified U,RNP. RNA The
sera
immunoprecipitation
sera
of the family members
were
subjected to RNA
immunoprecipitation and the results are seen in Figure 1. Sister Ju’s serum in all samples precipitated UIRNA, and two representative samples are seen in lanes 6 (9 August 1986) and 7 (19 May 1989). The first sample of Sister Je (lane 4) may immunoprecipitate a faint U,I~~~ band but the 19 May 1989 sample (lane 5) in3rnun~~ire~ipitat~s an intense U ¡RNA band. Lanes 8 m ELISA values for members’ sera.
anti-U1RNP
in
analysis
family
Idiotypic studies of the sisters’ ~r~~i-~I,.~.l~~’ and Fab fragments Rabbit serum 226 was prepared by immunization with Ju anti-UIRNP Fab. When absorbed with Cohn fil IgG, this did not react significantly with normal Fab2. as seen in Table III. In all cases, 5 Ag Fab fragments/ml were used to coat the plates and then the absorbed rabbit
Upper limit of normal values is 104 Ulml. Precipitation usually occurs with sera having at least 2 x 105 ~7‘~mt. Sister Ju’s anti-U¡RNP titer rose between November 1987 and May 1989 in association with development of Raynaud’s phenomenon.
252
Downloaded from lup.sagepub.com at NORTH CAROLINA STATE UNIV on March 18, 2015
Table IV Comparison of various anti-U,RNP coats in reaction with anti-idiotype (Id) reagent No. 226 in ELISA.
Anti-UIRNP antibodies
react strongly with anti-M reagent; antiRo/SSA and anti-La/SSB antibodies react poorly with angi-I~ reagents. Values listed are OD at 405 nm in the anti-Id ELISA.
Table V
HLA
profiles
on
family.
Figure 2 Western blot analysis with HeLa cell
extract and family members’ sera. Lane 1: normal human serum; lanes 2+3: antiRo/SSA and anti-La/SSB sera; lane 4: anti-nRNP serum; lane 5: mother’s sera in 1986; lane 6: mother’s sera in 1989; lane 7: father’s sera in 1986; lane 8: father’s sera in 1987; lane 9: father’s sera in 1989; lane 10: sister Ju’s sera in 1986; lane I1: sister Ju’s sera in 1987; lane 12: sister Ju’s sera in 1989; lane 13: sister Je’s sera in 1986; lane 14: sister Je’s sera in 1987; lane 15: sister Je’s sera in 1989. Sister Je has anti-68 kDa in all sera. Sister Ju has anti-65 kDa in all sera but makes anti-68 kDa in 1989 sample. Mother has antibody to a 75-kDa protein in HeLa
been previously associated with the several studies 8-11.
extract.
D R4DQw3
alleles in
o
Discussion
added at dilutions of 10-2 and 10 -’. After washing, goat anti-rabbit IgG alkaline phosphatase was added. After appropriate incubation and washing, paranitrophenyl-phosphate (substrate) was added and the OD values at 405 nm read at 1 h with a Multitek serum was
are several points that make the study of this family notable. Much has been made c~:~ the relationship
There
of antibodies to U,RNP and its 68-kDa polypeptide and the syndrome of MCTD. Indeed, sister Je had polymyositis and features of scleroderma (sclerodactyly, small oral aperture and Raynaud’s phenomenon). The second sister, however, did not satisfy criteria for any disease but exhibited a grand mal seizure disorder and Raynaud’s phenomenon. Her clinical picture has been stable; her seizure disorder is well controlled with anticonvulsant medications and her Raynaud’s phenomenon has been treated with procardia with moderate success. Thus, their very similar autoimmune are associated with quite different clinical pictures, except that they both manifest ~.a~naud’~ phenomenon. Such data that, except for Raynaud’s phenomenon, there is no mechanistic relationship between clinical expression of disease and antibodies to ~.1~~.I~I~’. Finally, the presence of substantial titers of unrelated autoantibodies in the two parents raises anew the question of the significance of such specificities in asymptomatic first-degree relatives. An ELISA study probing the relationship of genetic factors in the major histocompatibility complex and specific autoantibodies in asymptomatic first-degree, relatives in SLE and Sj6gren’s
scanner.
As seen, all anti-UtRNP Fabs tested were reactive, but the sisters’ UiRNP Fab fragments were more antigenic than those from other patients. Anti-Ro/SSA and anti-La/SSB Fab fragments were non-reactive. Each sister was slightly superior ~~t~~~~i~~lly to the other sister when the serum prepared from their individual anti-U,.RNP Fab fragments were used to react with plates coated with their c~~~ ~,~.~~ Fab. This suggests that there are idiotypic determinants characteristic and cross-reactive of the anti-UIRNP specificity but also individually specific determinants by the two
sisters. HLA testing The results of HLA testing of the lymphocytes of the family members are listed in Table IV. As shown, the sisters were non-identical but shared the ~i~ss I alleles ~~~~~-, and the class 11 alleles DR.DQw3’ which they received from the mother. Antibodies to ~J,i~~fi~ have
253
Downloaded from lup.sagepub.com at NORTH CAROLINA STATE UNIV on March 18, 2015
syndrome families showed that ~5~0 of these relatives were anti-Ro/SSA positive and 90% had either DR2 or DR3 alleles 12. Thus, the same HLA-D alleles associated with high levels of anti-Ro/SSA in patients were associated with low but elevated levels of anti-Ro/SSA in first-degree relatives. Whether this confers a greater risk for the development of disease in these relatives remains to be seen. At least it can be concluded that autoantibody production in first-degree relatives has, in part, a genetic basis. We have not found the presence of antinucleolar antibodies in the parents of other patients who produce anti-U,RNP (or for that matter other autoantibodies such as anti-Sm, anti-Ro/SSA etc. in the lupus spectrum). In our families, about 25% of firstdegree asymptomatic relatives have a positive ANA test but none thus far with a nucleolar pattern. We have, however, found some other first-degree relatives as well as some SLE patients who also produce antibodies to a 75-kDa protein as did the mother and sister Je. Studies are underway to identify the molecular nature of this antigenic target to see what insights this information could reveal about the mechanism of autoimmune disease. The most striking finding in this family was the development of Raynaud’s phenomenon in sister Ju coincident with a 20-fold increase in the titer of anti-U IRNP. While recent studies of patient populations have shown a strong association of Raynaud’s phenomenon and anti-U1RNp2-4, this is the first reported example of a temporal relationship of a large increase of anti-UIRNP and the de novo development of Raynaud’s phenomenon. This suggests that anti-U1RNP itself or an associated antibody or other associated biological mediator is involved in the pathogenesis of Raynaud’s
most important lesson from this family is that there is much to be learned from long-term study studies that permit the observation of the transifamily tions from asymptomatic relative to patient that will occur in families with autoimmune diseases. The study of such families should improve our understanding of the relationship between genes and autoantibody production on the one hand and between autoantibodies and clinical expression on the other.
Perhaps the
Acknowledgements This work was supported by NIH grants POI A-21568 and ROt .P~~-31133.
References 1. Reichlin M.
2.
3.
4.
5.
6.
7.
8.
phenomenon. It is also quite interesting that, while the HLA profile of the two sisters is not identical, the autoimmune response was quite similar. The titers in both ELISA and ANA tests reflecting the antibody to U1RNP were quite similar, as were the RNAs immunoprecipitated and the proteins bound in Western blot. Even the idiotypic fine specificity of their anti-U,~I~T~ molecules was closely related, similar and distinctive amino acid sequences in the V region of either of their heavy and light chains or both. Since the two sisters shared the haplotype A2B27C2DR4DQW3, it appears that the DR, and/or the DQW3 play a crucial role in determining the precise molecular character of the autoimmune response.
9.
10.
11.
12.
Systemic lupus erythematosus.
In:
Bigazzi PE,
Reichlin M, eds. Systemic A utoimmunity. New York, Basel, Hong Kong: Marcel Dekker Inc., 1991: 163-99. Habets JW, De Rooij DJ, Salden MH et al. Antibodies against distinct nuclear matrix proteins are characteristic for mixed connective tissue disease. Clin Exp Immunol 1983 ; 54: 265-76. Petterson I, Wang G, Smith EI et al. The use of immunoblotting and immunoprecipitation of (U) small nuclear ribonucleoproteins with mixed connective tissue disease and systemic lupus erythematosus. Arthritis Rheum 1986; 29: 986-96. Reichlin M, Van Venrooij WJ. Autoantibodies to the URNP particles: relationship to clinical diagnosis and nephritis. Clin Exp 1991; 83: 286-90. Immunol Maddison PJ, Provost TT, Reichlin M. Serological findings in patients with ’ANA-negative’ systemic lupus erythematosus. Medicine 1981; 60: 87-94. Kulick K, Reichlin M. Antibodies to single-stranded DNA in patients with discoid lupus erythematosus. Arthritis Rheum 1982; 25: 639-46. Itoh Y, Kriet JD, Reichlin M. Organ distribution of the Ro/SSA antigen in the guinea pig. Arthritis Rheum 1990; 33: 1815-21. Nishikai M, Sekiguchie S. Relationship of autoantibody expression and HLA phenotype in Japanese patients with connective tissue disease. Arthritis Rheum 1985; 28: 579-81. Smolen JS, Klippel JA, Penner E et al. HLA DR antigens in systemic lupus erythematosus: association with specificity of autoantibody responses to nuclear antigens. Ann Rheum Dis 1987; 46: 457-62. Harley JB, Sestak AL, Willis LG, Fu SM, Hausen JA, Reichlin M. A model for disease heterogeneity in systemic lupus erythematosus. Arthritis Rheum 1989; 32: 826-36. Olsen ML, Arnett FC, Reveille JD. Anti Sm and RNP antibodies are associated with distinct and different DQa and DQβ genes. Arthritis Rheum 1990; 33: 100 (abstract B169). Arnett FC, Hamilton RG, Reveille JD, Bias WB, Harley JB, Reichlin M. Genetic studies of Ro(SS-A) and La(SS-B) autoantibodies in families with systemic lupus erythematosus and
primary Sjögren’s syndrome.
Arthritis Rheum 1989; 32: 413-9.
(Received 3 March 1992) (Accepted 17 April 1992)
254
Downloaded from lup.sagepub.com at NORTH CAROLINA STATE UNIV on March 18, 2015
