2002 Nature Publishing Group All rights reserved 1466-4879/02 $25.00 ... The immunoglobulin kappa (Km) light chain gene is polymorphic and is believed to ...
Genes and Immunity (2002) 3 (Suppl 1), S63–S65 2002 Nature Publishing Group All rights reserved 1466-4879/02 $25.00 www.nature.com/gene
Immunoglobulin kappa light chain gene alleles are not associated with primary Sjo¨gren’s syndrome S Downie-Doyle1, S Lester1, P Bardy2, T Gordon3, M Rischmueller1 and K Pile1 1
Department of Rheumatology, The Queen Elizabeth Hospital, Woodville, South Australia; 2Australian Red Cross Blood Services, Adelaide, South Australia; 3Department of Immunology, Allergy and Arthritis, Flinders Medical Centre, Bedford Park, South Australia
The immunoglobulin kappa (Km) light chain gene is polymorphic and is believed to play a role in the pathology of infectious and autoimmune diseases. Polymorphisms within the constant region of the Km gene encode three alleles designated Km1, Km1,2 and Km3. Previous studies using serological detection of Km allotypes reported associations between specific Km allotypes, systemic lupus erythematosus and the presence of anti-La antibodies, yet these findings were not confirmed in other studies. In order to more precisely define any associations between Km alleles and anti-Ro/La antibodies we used the polymerase chain reaction and restriction fragment length polymorphisms for Km genotyping in a large cohort of patients with primary Sjo¨gren’s syndrome (SS). No associations were observed between specific Km alleles and primary SS when compared with a control population, nor within serologically defined subsets of SS patients. We conclude that Km alleles are not associated with primary SS or the Ro/La autoantibody response. Genes and Immunity (2002) 3 (Suppl 1), S63–S65. doi:10.1038/sj.gene.6363853 Keywords: immunoglobulins; Sjo¨gren’s syndrome; Km allotypes; polymorphism
Primary Sjo¨gren’s syndrome (SS) is a systemic autoimmune disorder characterised by the presence of serum autoantibodies directed to the ubiquitous intracellular autoantigens Ro (SS-A) and La (SS-B).1 Although it shares a number of clinical and serological features with systemic lupus erythematous (SLE), the underlying aetiology and pathogenetic mechanisms in SS are largely unknown. A number of regions on genome-wide scans have been identified as conferring susceptibility to human autoimmune diseases such as SLE and diabetes mellitus and also to murine models of these diseases. One of the most important genetic loci in this respect is the major histocompatibility locus, encoding the human leukocyte antigens (HLA). In the absence of genome-wide scans in SS and a paucity of family studies, few candidate susceptibility loci have been identified apart from the HLA region.2,3 HLA class II genes are of critical importance in determining patterns of autoantibody production in SS patients: the DR2-DQ1 haplotype is highly predictive of anti-Ro antibodies either alone (OR 7.5, P ⬍ 0.01) or in conjunction with non-precipitating anti-La antibodies detected by ELISA only (OR 16.6, P ⬍ 0.001), whereas the DR3-DQ2 haplotype is associated with precipitating anti-Ro and anti-La antibodies detectable by counterimmunoelectrophresis (OR 47.1, P ⬍ 0.001).4 The latter Correspondence: M Rischmueller, Department of Rheumatology, The Queen Elizabeth Hospital Campus, North Western Adelaide Health Service, Woodville, South Australia, 5011 Australia. E-mail: Maureen.Rischmueller얀nwahs.sa.gov.au This study was supported by an Arthritis Foundation of Australia Val Provis Grant. Received 9 October 2001; revised and accepted 16 January 2002
subset of patients, in addition to having more diversified humoral responses to Ro and La, are also characterised by the presence of significantly higher levels of serum immunoglobulins and rheumatoid factor, suggesting that the development of humoral autoimmune responses might also be influenced by genes involved in the regulation of antibody production. The immunoglobulin kappa (Km) light chain gene locus is polymorphic and is believed to play a role in the pathology of infectious and autoimmune diseases.5 Within the constant region gene of the Km locus on chromosome 2 (2p12), polymorphisms have been identified which produce three Km alleles designated Km1, Km1,2, and Km3.5 Initially serologically defined, these polymorphisms result in specific amino acid differences at positions 153 and 191 of the constant region of the kappa polypeptide chain: Km1 has valine at position 153 and leucine at position 191; Km1,2 has alanine at position 153 and leucine at position 191; Km3 has alanine at position 153 and valine at position 191.6 These polymorphisms could potentially play a role in the pathology of autoimmune responses by indirectly influencing autoantibody specificity for autoantigens7 or alternatively they could be in linkage disequilibrium with one or several nearby autoimmune susceptibility genes. Serologically determined frequencies of the Km(1) allotype include both the Km1 and Km1,2 alleles, and as the Km1 allele is extremely rare, these frequencies represent Km1,2 alleles almost exclusively.6 Serologically determined Km(1) was initially reported to be associated with specific immune responses to bacterial polysaccharides8 and with IgA nephropathy9 and atopy.10 Subsequent serologically-based studies linked Km(1) with SLE11,12 and anti-La responses,13 whereas others did not confirm
Immunoglobulin allotypes in Sjo¨gren’s syndrome S Downie-Doyle et al
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Table 1 Allele frequencies of the Kappa (Km) gene in patients with primary Sjo¨ gren’s syndrome (pSS)a and controls Km 1,2 allele
Km 3 allele
All SS patients (n = 109) pSS Seronegativec (n = 19) pSS isolated Roc (n = 11) pSS Ro + La (ppt−)c (n = 21) pSS Ro + La (ppt+)c (n = 58)
0.09 0.11 0.05 0.07 0.10
0.91 0.89 0.95 0.93 0.90
Controls (n = 164)
0.08
0.92
No significant differences were detected within primary SS patient groups or between patients with primary SS and controls using the Population Differentation Test.17 a The patient group comprised 109 patients who fulfilled at least four of the six European Consensus Criteria for pSS.1 b The control population comprised healthy individuals from the unrelated Australian Bone Marrow Donor Registry, and were assumed to be a representative cross section of the South Australian population. c Anti-Ro and anti-La antibodies were characterised by counterimmunoelectrophoresis and by ELISA using native and recombinant antigens.19 Detection of Ro refers to the presence of precipitating anti-Ro antibodies either in the absence of anti-La (pSS isolated Ro); in association with non-precipitating anti-La antibodies detectable by ELISA only (pSS Ro + La precipitin negative (ppt−)); or in association with precipitating anti-La antibodies (pSS Ro + La precipitin positive (ppt+)).
these findings.14,15 More recently Km(3) was reported to be associated with an increased risk of SLE.16 These conflicting results may be explained by genetic differences between patient groups, or methodological problems associated with serological typing. On the basis of the above association studies we hypothesised that diversification of autoantibody responses to Ro and La might be under the influence of polymorphisms within the constant region of the Km gene. The aims of this study were to (i) compare the frequency of Km1, Km1,2, and Km3 polymorphisms between a cohort of 109 patients with primary SS and 164 controls, and (ii) determine whether specific Km alleles are associated with diversification of autoantibody responses to Ro and La. Polymerase chain reaction (PCR) was used to amplify a 361-bp product, as previously described,7 followed by restriction fragment length polymorphism (RFLP) using sequential digestion by Acc I and Tai I then separation on a 2% agarose gel. The technique was verified using a combination of direct sequencing and testing of reference DNA samples. Statistical comparison used the Population Differentiation Test.17 The Km3 allele was identified by a polymorphic site at position 191 (C to G) which resulted in 113 bp and 248 bp products following Acc I digestion. Completed digestion when using the Acc I enzyme required 55°C incubation. Potential Km1,2 or Km 1 alleles were identified as uncut 361 bp products following Acc I digest. The Km1 allele, with a polymorphic site at position 153 (C to T), resulted in 131 bp and 230 bp products following Tai I digestion, whereas lack of digestion indicated the Km1,2 allele. The Km1,2 allele was detected in both the control and patient groups (0.08 and 0.09 respectively) as was the Km3 allele (0.92 and 0.91 respectively), but not the rare Km1 allele (Table 1). The Km genotype distribution in both the SS patient and control groups were in Hardy-Weinberg equilibrium (P = 0.976, 0.993 respectively). The allele frequencies were similar to those previously reported in other populations using PCR/RFLP methods.7,18 To summarise the findings in this study, no association was seen between any specific Km allele and primary SS when compared with the control population, nor with the following serologically defined subsets of SS patients: (i) seronegative (no anti-Ro/La antibodies, n = 19), (ii) isoGenes and Immunity
lated anti-Ro antibodies (n = 11), (iii) anti-Ro and nonprecipitating anti-La (ppt−, n = 21), (iv) anti-Ro and precipitating anti-La (ppt+, n = 58). A study of Km allotypes in SLE using PCR-based methodology reported similar frequencies to our study in patient and control groups: 0 and 0.005 respectively for Km1; 0.08 and 0.10 respectively for Km1,2; and 0.92 and 0.90 respectively for Km3.18 In conclusion, in contrast to previous smaller studies of autoimmune diseases using serological methods, our larger controlled study using PCR/RFLP revealed no associations between any of the three Km alleles and primary SS nor its serologic subsets. Km alleles do not appear to be important in controlling the diversification of autoantibody responses to Ro and La.
Acknowledgements Special thanks to Dr G Moxley, Medical College of Virginia, Richmond, USA who kindly donated reference DNA samples. This work was supported by the Australian Red Cross Blood Service and the Arthritis Foundation of Australia Val Provis Grant. We thank members of the Arthritis Foundation of South Australia Lupus/Scleroderma/Sjo¨ gren’s support group, and other Sjo¨ gren’s syndrome sufferers for their involvement.
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