Presence of apolipoprotein E {epsilon}4 allele predisposes to early onset of primary Sjögren's syndrome

M. Pertovaara, T. Lehtimäki1, R. Rontu1, J. Antonen1, A. Pasternack1 and M. Hurme1

Tampere University Hospital and 1 University of Tampere Medical School and Tampere University Hospital, Tampere, Finland.

Correspondence to: M. Pertovaara, Department of Internal Medicine, Section of Rheumatology, Tampere University Hospital, P.O. Box 2000, FIN-33521 Tampere, Finland. E-mail: marja.pertovaara{at}pshp.fi


    Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 References
 
Background. Apolipoprotein E (apoE) polymorphism plays a central role in lipid metabolism, but has recently also been suggested to regulate inflammation, as judged by levels of serum C-reactive protein (CRP).

Objective. To establish whether polymorphism of the apoE genes affects susceptibility to primary Sjögren's syndrome (pSS), degree of inflammation or age of onset of pSS.

Methods. ApoE genotype distribution and allelic frequencies were analysed using PCR and the TaqMan system in 63 Finnish Caucasian patients with pSS and in 64 healthy controls matched for sex, ethnic origin and area of residence. The clinical and immunological data on the pSS patients were analysed in relation to the apoE genotypes.

Results. There was no difference between pSS patients and controls in apoE genotype and allelic frequencies. The apoE {epsilon}4 allele was significantly associated with early onset of pSS in the entire population and in female patients (Kaplan–Meier log rank test, P = 0.0407 and P = 0.0168, respectively). The average age (± S.D.) of onset of pSS in all apoE {epsilon}4 allele carriers was 46 ± 12 and in other genotypes it was 53 ± 10 yr (P = 0.031, t-test). ApoE polymorphism was not associated with signs of inflammation evaluated by such markers as concentration of plasma CRP, plasma interleukin-6, plasma TNF-{alpha}, immunoglobulin G and haemoglobin, or leucocyte count or ESR.

Conclusions. ApoE polymorphism does not affect susceptibility to pSS or levels of plasma inflammatory indices in patients with pSS. However, a clear association prevails between apoE {epsilon}4 and early onset of pSS.

KEY WORDS: Apolipoprotein E, Gene polymorphism, Primary Sjögren's syndrome, Age at disease onset


    Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 References
 
Primary Sjögren's syndrome (pSS) is a chronic inflammatory autoimmune disease characterized by symptoms of dry eyes and mouth, various extraglandular symptoms, hypergammaglobulinaemia and abundant autoantibody production. Plasma levels of interleukin-6 (IL-6), a proinflammatory cytokine, are increased in patients with pSS [1].

Apolipoprotein E (apoE) gene polymorphism plays a significant role in lipid metabolism, but in addition has recently also been suggested to regulate inflammation, as measured by levels of C-reactive protein [2]. The gene for apoE is polymorphic, presenting with three common alleles ({epsilon}2, {epsilon}3, {epsilon}4) at a single gene locus on chromosome 19. These alleles form six genotypes: E2/2, E3/2, E4/2, E3/3, E4/3 and E4/4, of which E3/3 is the commonest and E4/3 the second commonest in most populations. The presence of the apoE {epsilon}4 allele is associated with an increased risk of coronary heart disease and Alzheimer's disease [reviewed in 3].

Circulating testosterone and dehydroepiandrosterone (DHEA) levels have recently been found to be associated with the apoE genotype and women carrying the apoE {epsilon}4 allele thus to be more susceptible to the development of some diseases associated with the menopause [4]. Sjögren's syndrome presents characteristically in middle-aged women. We hypothesized that apoE could be a candidate gene for susceptibility to pSS and, further, that the genetic polymorphism of apoE could exert an effect on markers of inflammation in patients with pSS. Hence, the aims of the present study were to investigate in a well-characterized group of Finnish Caucasian patients with pSS the effect of the polymorphism of apoE gene on susceptibility to and severity of pSS, particularly on markers of inflammation and age of onset of pSS.


    Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 References
 
Subjects
All patients fulfilling three or more modified Californian criteria for pSS [5] (salivary flow determinations were not performed; histological findings were graded on the Chisholm–Mason [6] scale, grades 3 and 4 being regarded as diagnostic) were selected from the records of patients with sicca symptoms examined in the Department of Internal Medicine, Section of Rheumatology, at Tampere University Hospital, Finland, during the years 1977 to 1992 (n = 111). Those alive were invited by letter to attend for gene polymorphism determinations, and samples for genotyping were obtained after informed consent from 63 pSS patients (61 female, two male, mean age 60 ± 12 yr, mean disease duration 9 ± 4 yr). Sixty-one of them also fulfilled the revised American–European consensus group criteria for pSS [7].

Clinical methods
A careful clinical examination together with an in-depth interview with the patients, covering previous diseases, duration of sicca symptoms and existence of recurrent salivary gland swellings, had recently been conducted with these patients [8]. Special emphasis was laid on possible extraglandular symptoms of pSS (dermatological, endocrine, gastrointestinal, lymphoproliferative, musculoskeletal, neurological, renal, respiratory and vascular symptoms).

Standard laboratory tests
The standard laboratory tests included blood cell count, erythrocyte sedimentation rate (ESR) and plasma C-reactive protein (CRP). Anti-SS-A and anti-SS-B antibodies were determined by enzyme immunoassay. Serum concentrations of immunoglobulin G (IgG) were measured by laser nephelometry and serum ß2-microglobulin by radioimmunoassay (Pharmacia beta-2-micro RIA kit; Pharmacia Diagnostics, Uppsala, Sweden). Plasma IL-6 and TNF-{alpha} concentrations were determined using commercially available ELISAs (Pelikine human IL-6 ELISA kit and Pelikine Compact human TNF-a ELISA kit; CLB, Amsterdam, The Netherlands). The optical density of individual wells was determined with a Multiscan Biochromatic 348 spectrophotometer (Thermo Labsystems, Helsinki, Finland). The detection limit of the IL-6 assay was 0.6 pg/ml and that of the TNF-{alpha} assay 1.4 pg/ml.

Normal controls
Sixty-four healthy Finnish Red Cross Transfusion Service blood donors matched for sex (62 female, two male), ethnic origin (Finnish Caucasian) and area of residence (Tampere, Finland) served as a control group for DNA studies of pSS patients. Mean age of the control subjects was 53 ± 7 yr.

Apolipoprotein E genotyping
Genomic DNA was extracted from peripheral blood leucocytes using a commercially available kit (Qiagen, Hilden, Germany). For the APOE 112 genotyping, fluorogenic allele-specific TaqMan probes and primers were used as previously described [9]. APOE 158 genotypes were determined using allele-specific fluorogenic probes with a conjugated minor groove binder (MGB) group. The nucleotide sequences of the APOE 158 primers and probes used in the PCR were deduced from published sequences deposited in the GenBank database and were chosen and synthesized in conjunction with Applied Biosystems (Foster City, CA, USA) using the Assay-by-Design tool. DNA samples were genotyped by means of the 5' nuclease assay for allelic discrimination using the ABI Prism 7000 Sequence Detection System (Applied Biosystems). A polymerase chain reaction (PCR) reaction containing genomic DNA, 1 x Universal PCR Master Mix, 900 nM of each primer and 200 nM of each probe was performed in 96-well plates using the standard protocol in a total volume of 25 µl. Water controls and known control samples previously typed by restriction fragment length polymorphism–PCR analysis were run in parallel with unknown DNA samples. After cycling, end-point fluorescence was measured and genotype calling carried out using the allelic discrimination analysis module.

Statistical analysis
Unpaired Student's t-test and {chi}2 test were used in comparisons of continuous and dichotomous variables, respectively. The association of the {epsilon}4 allele with age at onset of pSS was analysed with the Kaplan–Meier log rank and t-tests. Findings were considered statistically significant at P<0.05. Statistical analyses were performed with SPSS 10.1 for Windows (SPSS, Chicago, IL, USA).

Ethical approval
The study protocol was approved by the Ethical Committee of Tampere University Hospital.


    Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 References
 
ApoE genotypes and allelic frequencies and age of onset of pSS
The apoE genotype and allele frequencies in pSS patients and control subjects did not differ (Table 1). However, pSS patients carrying the apoE {epsilon}4 allele were significantly younger (mean ± S.D.) (55 ± 11 vs 62 ± 11 yr, P = 0.028) than non-carriers. Moreover, the apoE {epsilon}4 allele was associated with earlier onset of pSS in the entire population and in female patients (Kaplan–Meier log rank test, P = 0.0407 and 0.0168, respectively) (Fig. 1). The average age at disease onset in all apoE {epsilon}4 allele carriers was 46 ± 12 yr and in other genotypes it was 53 ± 10 yr (P = 0.031, t-test and P = 0.039 for the patients fulfilling the American–European consensus group criteria, t-test). The mean age of female patients with pSS carrying the apoE {epsilon}4 allele was 54 ± 11 yr compared with 62 ± 11 yr in non-carriers (P = 0.023), and their age at disease onset was 45 ± 12 vs 53 ± 10 yr (P = 0.019), respectively.


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TABLE 1. Apolipoprotein E (apoE) genotype and allelic frequencies in patients with primary Sjögren's syndrome (pSS) and in healthy subjects

 


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FIG. 1. Kaplan–Meier plots showing the earlier age at onset in (a) both female and male patients with primary Sjögren's syndrome (pSS) carrying the {epsilon}4 allele of the apolipoprotein E gene (P = 0.0407, log rank test) and (b) female patients with pSS carrying the {epsilon}4 allele of apolipoprotein E gene (P = 0.0168, log rank test).

 
ApoE polymorphism and markers of inflammation
The mean concentration of plasma CRP of the pSS patients was 5.8 ± 3.2 mg/l, that of plasma IL-6 4.0 ± 2.6 pg/ml and that of plasma TNF-{alpha} 7.1 ± 13.4 pg/ml. There were no statistically significant differences in such markers of inflammation as plasma CRP levels (6.5 ± 4.2 vs 5.5 ± 2.8 mg/l), plasma IL-6 (3.2 ± 2.6 vs 4.1 ± 2.6 pg/ml) and plasma TNF-{alpha} concentrations (7.4 ± 13.1 vs 7.0 ± 14.0 pg/ml) between pSS patients carrying the apoE {epsilon}4 allele and those not carrying this allele. Haemoglobin concentration (128 ± 7 vs 128 ± 13 g/l), leukocyte count (5.09 ± 2.58 x 109/l vs 5.12 ± 1.56 x 109/l), ESR (35 ± 20 vs 30 ± 20 mm/h), serum IgG (20.6 ± 7.7 vs 18.0 ± 6.8 g/l) and serum ß2-microglobulin concentrations (2.90 ± 1.03 vs 2.90 ± 1.06 mg/l) did not differ between these patient groups. Neither did anti-SSA (78 ± 45 vs 64 ± 55 IU/l) and anti-SSB antibody titres (56 ± 70 vs 67 ± 76 IU/l) differ between apoE {epsilon}4 carriers and non-carriers.

ApoE polymorphism and clinical features of pSS
No differences were observed in the frequencies of such extraglandular manifestations of pSS as a history of arthralgia, arthritis, salivary gland swelling, Raynaud's symptom, purpura, lymphadenopathy, pleuritis, alveolitis or pulmonary fibrosis, or peripheral or central nervous system symptoms in patients with or without apoE {epsilon}4 allele. The number of diagnostic criteria for pSS did not differ between these patient groups (data not shown). The histological grade in labial salivary gland biopsies was lower in apoE {epsilon}4 carriers than in non-carriers (grade 3–4 in 56 vs 80%, P = 0.048, respectively).

Discussion
Genetic variation of the apoE gene influences susceptibility to coronary heart disease and sporadic Alzheimer's disease, apoE {epsilon}4 being the allele with the strong association. Recently, genetic variation in the apoE gene was also found to affect the degree of inflammation by influencing serum CRP levels in dyslipidaemic middle-aged men [2]. Surprisingly, however, it was the patients carrying the apoE {epsilon}4 allele, known to be associated with inflammation, who were found to have lower CRP levels than the others [2].

In patients with pSS, chronic inflammation is present, as shown by markedly elevated ESR levels, pronounced hypergammaglobulinaemia and elevated IL-6 levels. We therefore hypothesized that genetic polymorphism of the apoE could have an effect on markers of inflammation in patients with pSS. However, this proved not to be the case: first, no differences were found in the apoE genotype or allele frequencies between pSS patients and healthy controls. Moreover, the apoE {epsilon}4 allele did not influence the levels of CRP in patients with pSS. This finding might be explained by the fact that pSS is known to belong to a group of autoimmune diseases with a modest or even absent CRP reaction [10]. However, in addition to lack of differences in CRP levels, no differences emerged in any other well-established markers of inflammatory activity, including plasma IL-6, TNF-{alpha} and IgG between apoE {epsilon}4 carriers and non-carriers. The apoE {epsilon}4 carrier state was also not associated with clinical features of the disease. Labial salivary gland histology, a marker of severity of inflammation in pSS, was even milder in apoE {epsilon}4 allele carriers compared with non-carriers.

We found that the age at disease onset was significantly lower in pSS patients with the apoE {epsilon}4 allele than in those without this allele. Similarly, it has been noted that the effect of the apoE {epsilon}4 allele on coronary atherosclerosis [11] and Alzheimer's disease [12] is age-dependent. Moreover, in recent studies, apoE polymorphism has also been found to affect the age at onset of infectious diseases [13, 14]. ApoE polymorphism was found to affect the age of becoming infected with Plasmodium falciparum in children from Ghana [13] and to have a possible role in protection against the early stages of malaria [14]. Viral infections, e.g. Epstein–Barr, cytomegalovirus or retrovirus infections have been proposed to act as triggering agents in the development of pSS in genetically susceptible subjects [15]. There are no data on apoE polymorphism and susceptibility to or age at disease onset related to these infections. We do not know the background to the significant association between apoE {epsilon}4 and earlier disease onset of pSS. One might speculate, however, that this association could be mediated, for example, through a possible effect on the time-point of a triggering viral infection.

A further speculative explanation for the earlier age at onset of pSS, particularly in female subjects carrying the apoE {epsilon}4 allele, might be a relation to hormonal factors, which in view of the strong female predominance among patients with pSS are also believed to be involved in the pathogenesis of pSS [15]. The gender bias and the concomitant characteristic late age of disease onset in pSS have been difficult to interpret, as oestrogens have been considered to favour autoimmunity and androgens to protect from it. Carriers of apoE {epsilon}4 have been held to be more susceptible to the development of some diseases associated with menopause, as they have been found to have higher serum DHEA and testosterone levels than women not carrying this allele [4]. Furthermore, an interaction between oestrogen and apoE in the Alzheimer's disease process has been observed; in a recent study apoE polymorphism influenced binding to the oestrogen receptor and altered transcriptional activity in response to oestrogen [16].

We have demonstrated that apoE polymorphism does not affect susceptibility to pSS or plasma inflammatory indices in patients with pSS, but there is a clear association between apoE {epsilon}4 allele and early onset of pSS.


    Acknowledgments
 
This study was supported by the Medical Research Fund of Tampere University Hospital, Tampere, Finland and the Maud Kuistilas Foundation.

The authors have declared no conflicts of interest.


    References
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 Abstract
 Introduction
 Subjects and methods
 Results
 References
 

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Submitted 2 June 2004; revised version accepted 23 July 2004.



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