Prevalence of STAT3 mutations in patients with rheumatoid arthritis-associated T-cell large granular lymphocytic leukaemia and Felty syndrome.

OBJECTIVES: Neutropenia is a key presentation of Felty syndrome (FS) and rheumatoid arthritis (RA)-associated T-cell large granular lymphocytic (T-LGL) leukaemia. Clonal rearrangement of T-cell receptor (TCR) gene supports the diagnosis of T-LGL leukaemia but not FS. Mutations in the signal transducer and activator of transcription 3 (STAT3) gene are highly specific for T-LGL leukaemia, but their prevalence in FS remains poorly clarified. METHODS: The study included 100 patients with RA and unexplained neutropenia. TCR rearrangements were examined in blood (100 cases), bone marrow (47 cases), and spleen (12 cases) using the BIOMED-2 protocol. Patients were stratified into RA-associated T-LGL leukaemia cohort if a clonal TCR rearrangement was identified in any of the tested patient samples, and into FS cohort in other cases. Mutations in the STAT3 were examined using next-generation sequencing (NGS) technology in blood (100 cases), bone marrow (37 cases), and spleen (7 cases). RESULTS: STAT3 mutations were identified in 71% (49/69) patients with RA-associated T-LGL leukaemia and in 10% (3/31) patients with FS (p=4.7×10-8). Three samples from the RA-associated T-LGL leukaemia cohort and 5 samples from the FS cohort had STAT3 mutations in the absence of clonal TCR rearrangement. CONCLUSIONS: The results suggest that STAT3 mutations are significantly less common in FS than in RA-associated T-LGL leukaemia. Moreover, NGS can detect clones undetectable by fragment analysis. We speculate that in patients with RA and neutropenia, the detection of STAT3 mutations can point to T-LGL leukaemia even in the absence of clonal TCR rearrangement.

The spectrum of somatic mutations in large granular lymphocyte leukemia, rheumatoid arthritis, and Felty's syndrome.

T cell large granular lymphocyte leukemia (T-LGLL) is an interesting case at the intersection of autoimmunity and cancer. In T-LGLL, T cells with somatic pathogenic mutations (mainly in STAT3) are linked to rheumatoid arthritis (RA) and neutropenia. A rare subtype of RA, Felty's syndrome, exhibits overlapping clinical features and comparable frequencies of activating STAT3 mutations in T cells as T-LGLL, which hints at a potential T-LGLL-Felty's syndrome-RA axis. Somatic mutations could shed light on the unexplained pathologies of these disorders. However, the causality of somatic mutations-do somatic mutations in immune cells cause inflammation, or does prolonged inflammation predispose to mutagenesis-remains unanswered. This review will focus on the recent advances in understanding somatic mutations in T-LGLL and related autoimmune conditions as a master regulatory network that sustains lymphoproliferation and inflammation.

Analysis of a single-institution cohort of patients with Felty's syndrome and T-cell large granular lymphocytic leukemia in the setting of rheumatoid arthritis.

T-cell large granular lymphocytic leukemia (T-LGLL) is a lymphoproliferative disorder characterized by a persistent increase in the number of large granular lymphocytes (LGLs), neutropenia, and splenomegaly. Clinical manifestations of T-LGLL in the setting of rheumatoid arthritis (RA) are often identical to those in which one would suspect Felty's syndrome (FS). These disorders are distinguished by the presence of T-cell clonality, which is present in T-LGLL but not in FS. Mutations in the signal transducer and activator of transcription 3 (STAT3) and 5b (STAT5b) genes can be used as molecular markers of T-LGLL, but their prevalence in FS is unknown.Eighty-one patients with RA and unexplained neutropenia or/and an increase in the number of LGLs above 2 × 109/L were stratified into RA-associated T-LGLL (N = 56) or FS (N = 25) groups based on the presence or absence of T-cell clonality. STAT3 and STAT5b gene mutations were assessed in each group by means of allele-specific polymerase chain reaction assays. Clinical, immunological, laboratory data and the results of immunophenotyping of blood and bone marrow lymphocytes were also evaluated.Mutations of the STAT3 gene and an increase in the number of LGLs above 2 × 109/L were detected in RA-associated T-LGLL, but not in FS (39% vs 0% and 21% vs 0%, respectively). Mutations in the STAT5b gene were not observed in either group. Expression of CD57, CD16, and CD5-/dim on CD3+CD8+ T-lymphocytes was observed in both RA-associated T-LGLL and FS.STAT3 gene mutations or LGL counts over 2 × 109/L in RA patients are indicative of T-LGLL.

Phenotypic variability in a child with Felty's syndrome: a case report.

BACKGROUND: Felty's syndrome (FS) is characterized by the triad of rheumatoid arthritis (RA), splenomegaly and neutropenia. The arthritis is typically severe and virtually always associated with high-titer rheumatoid factor. The presence of persistent neutropenia is generally required to make the diagnosis. Most patients diagnosed with FS are aged 50-70 years and have had RA for more than 10 years. It is rarely seen in patients with juvenile idiopathic arthritis (JIA), with only five cases having been reported throughout the world. CASE PRESENTATION: The present study describes the case of a 14-year-old female with a seven-year history of polyarticular JIA, presenting with splenomegaly, hepatomegaly, cholestasis and thrombocytopenia. However, she occasionally developed neutropenia. Titers of rheumatoid factor and anti-CCP were persistently high, and the antinuclear antibody titer was 1:320, while the antibody results for anti-dsDNA and anti-Sm were negative. Serum levels of IgA, IgG, IgM and IgE were all persistently elevated, and the ratio of CD19+ lymphocytes in the subgroups of lymphocytes was persistently high. The level of complements was normal. No STAT3 and STAT5B mutations were found by next-generation sequencing. The patient did not respond to methotrexate, prednisolone, hydroxychloroquine (HCQ), sulfasalazine and etanercept but was responsive to rituximab. CONCLUSIONS: JIA, thrombocytopenia and splenomegaly are the most common and important features in six children with FS, while persistent neutropenia is not seen in all these patients. No complement deficiency has been found in children with FS so far. Manifestations of FS without neutropenia may be extremely rare. There are differences between adults and children in the clinical and laboratory features of FS.

Somatic STAT3 mutations in Felty syndrome: an implication for a common pathogenesis with large granular lymphocyte leukemia.

Felty syndrome is a rare disease defined by neutropenia, splenomegaly, and rheumatoid arthritis. Sometimes the differential diagnosis between Felty syndrome and large granular lymphocyte leukemia is problematic. Recently, somatic STAT3 and STAT5B mutations were discovered in 30-40% of patients with large granular lymphocyte leukemia. Herein, we aimed to study whether these mutations can also be detected in Felty syndrome, which would imply the existence of a common pathogenic mechanism between these two disease entities. We collected samples and clinical information from 14 Felty syndrome patients who were monitored at the rheumatology outpatient clinic for Felty syndrome. Somatic STAT3 mutations were discovered in 43% (6/14) of Felty syndrome patients with deep amplicon sequencing targeting all STAT3 exons. Mutations were located in the SH2 domain of STAT3, which is a known mutational hotspot. No STAT5B mutations were found. In blood smears, overrepresentation of large granular lymphocytes was observed, and in the majority of cases the CD8+ T-cell receptor repertoire was skewed when analyzed by flow cytometry. In bone marrow biopsies, an increased amount of phospho-STAT3 positive cells was discovered. Plasma cytokine profiling showed that ten of the 92 assayed cytokines were elevated both in Felty syndrome and large granular lymphocyte leukemia, and three of these cytokines were also increased in patients with uncomplicated rheumatoid arthritis. In conclusion, somatic STAT3 mutations and STAT3 activation are as frequent in Felty syndrome as they are in large granular lymphocyte leukemia. Considering that the symptoms and treatment modalities are also similar, a unified reclassification of these two syndromes is warranted.

The impact of HLA-DRB1 genes on extra-articular disease manifestations in rheumatoid arthritis.

The objective of this study was to examine HLA-DRB1 and HLA-DQB1 genotypes in patients with severe extra-articular rheumatoid arthritis (ExRA) and to compare them with the genotypes of rheumatoid arthritis (RA) patients without extra-articular manifestations. Patients with severe ExRA were recruited from a large research database of patients with RA, from two cohorts of prevalent RA cases, and from a regional multicenter early RA cohort. Cases with ExRA manifestations (n = 159) were classified according to predefined criteria. Controls (n = 178) with RA but no ExRA were selected from the same sources. Cases and controls were matched for duration of RA and for clinical center. PCR based HLA-DRB1 and HLA-DQB1 genotyping was performed using the Biotest SSP kit, with additional sequencing in order to distinguish DRB1*04 subtypes. Associations between alleles and disease phenotypes were tested using multiple simulations of random distributions of alleles. There was no difference in global distribution of HLA-DRB1 and HLA-DQB1 alleles between patients with ExRA and controls. DRB1*0401 (P = 0.003) and 0401/0401 homozygosity (P = 0.002) were more frequent in Felty's syndrome than in controls. The presence of two HLA-DRB1*04 alleles encoding the shared epitope (SE) was associated with ExRA (overall odds ratio 1.79, 95% confidence interval 1.04-3.08) and with rheumatoid vasculitis (odds ratio 2.44, 95% confidence interval 1.22-4.89). In this large sample of patients with ExRA, Felty's syndrome was the only manifestation that was clearly associated with HLA-DRB1*0401. Other ExRA manifestations were not associated with individual alleles but with DRB1*04 SE double dose genotypes. This confirms that SE genes contribute to RA disease severity and ExRA. Other genetic and environmental factors may have a more specific impact on individual ExRA manifestations.

Major histocompatility complex haplotypic associations in Felty's syndrome and large granular lymphocyte syndrome are secondary to allelic association with HLA-DRB1 *0401.

OBJECTIVE: To investigate the role of HLA class I in susceptibility to Felty's syndrome (FS) and large granular lymphocyte (LGL) syndrome. METHODS: Fifty caucasoid FS patients, and 55 patients with LGL syndrome, of whom 26 had arthritis and 29 did not, were studied. Complete HLA class I and HLA-DR typing including, where relevant, DRB1*04 subtyping was carried out by molecular methods. Comparison was made with 78 unselected healthy caucasoid controls and a further 29 DRB1*0401+ individuals. RESULTS: A significant association was found between HLA-A*02 and FS [odds ratio (OR) 3.9, 95% confidence interval (95% CI) 1.8-8.4, P = 0.0004]. At the B locus, there was an association between B*44 and LGL with arthritis [OR 3.5 (1.3-9.2), P = 0.01]. For HLA-Cw*0501, there was an association with FS [OR 4 (1. 7-9.2) P = 0.0008]. For both FS and LGL with arthritis, the extended haplotype HLA-A*02;B*44;Cw*0501;DRB1*0401 was significantly associated [OR 9.5 (2.6-35), P = 0.0001; OR 4.6 (1-22.4), P = 0.05, respectively]. There was no association between HLA class I or II and LGL without arthritis. All the allelic and haplotypic associations were lost on comparison with HLA-DRB1*0401+ controls. The strongest HLA association was with HLA-DRB1*0401 for FS [OR 27.9 (10.3-75.5), P = 10(-13)], and LGL with arthritis [OR 35.4 (9.6-131. 3), P = 10(-10)]. CONCLUSIONS: The major histocompatibility locus (MHC) associations with FS reported here are due to linkage disequilibrium with HLA-DRB1*0401. LGL syndrome with arthritis shows identical class II associations with FS, although there may be subtle immunogenetic differences between the two in the class I region. One of the extended haplotypes reported in a number of studies for FS and rheumatoid arthritis (summarized as HLA-A*02;Cw*0501; B*44;TNFb5;TNFa6;TNFd4;C4A*3;C4BQ*0;DRB1*0 401;DQB1*0301) is likely to be attributable to strong primary association with HLA-DRB1*0401, rather than to epistatic interaction between these loci.

Genetic polymorphism of IL-12 p40 gene in immune-mediated disease.

Understanding of the genetic basis of autoimmune diseases is currently incomplete. Cytokine gene polymorphisms warrant consideration as factors explaining variation in the human immune and inflammatory responses and as candidate susceptibility genes for related pathological states. Interleukin 12 (IL-12) is a key regulator of the polarisation of immune responses to T helper 1 or 2 categories and plays a role in autoimmune and infectious diseases. Using a bioinformatic strategy, we aligned cDNA and expressed sequence tag sequences to identify putative polymorphic regions of the IL-12 p40 gene. Position 1188 in the 3' untranslated region (UTR) was polymorphic with the frequency of the common allele around 80% in healthy UK Caucasoids. PCR genotyping of multiple Caucasoid groups and an African group showed significant population variation. In a case-control design, the polymorphism was not associated with rheumatoid arthritis, Felty's syndrome or large granular lymphocyte syndrome with arthritis or multiple sclerosis. A nonsignificant increase in the B allele frequency was observed in the rare large granular lymphocyte syndrome without arthritis (odds ratio 2.02 95% CI 0.95-4.3). This new genetic marker could be useful in anthropological studies and should be investigated in other autoimmune, allergic, inflammatory and infectious diseases.

A Fas promoter polymorphism at position -670 in the enhancer region does not confer susceptibility to Felty's and large granular lymphocyte syndromes.

OBJECTIVE: We examined whether there are associations between a polymorphism in the Fas promoter, recently found to be associated with rheumatoid arthritis (RA), and Felty's syndrome or large granular lymphocyte (LGL) leukaemia. METHODS: Thirty-five patients with Felty's were studied, along with 18 patients with LGL syndrome and arthritis, 17 patients with LGL syndrome but no arthritis, and 128 controls. The polymorphism was typed by polymerase chain reaction followed by digestion with the restriction enzyme MvaI. RESULTS: No significant difference was found in genotype or allele frequencies between the groups. CONCLUSION: This promoter polymorphism is not a significant risk factor responsible for the LGL expansions seen in Felty's and LGL syndromes. Abnormal, constitutive expression of Fas ligand may be more relevant to the aetiology of these diseases.

Interleukin-10 promoter polymorphisms in rheumatoid arthritis and Felty's syndrome.

OBJECTIVE: To examine whether promoter polymorphisms associated with variation in interleukin-10 (IL-10) production are relevant to the development of rheumatoid arthritis (RA) or Felty's syndrome (FS). METHODS: DNA was obtained from 44 FS patients, 117 RA patients and 295 controls. The promoter region between -533 and - 1120 was amplified by polymerase chain reaction, and polymorphisms detected by restriction enzyme digest or sequence-specific oligonucleotide probing. RESULTS: We found no significant difference in allele or haplotype frequencies between the groups. CONCLUSION: There is no association between FS or RA and these recently identified IL-10 promoter polymorphisms. Other genetic or environmental factors could explain the alterations in IL-10 levels seen in these conditions.

Immunogenetic similarities between patients with Felty's syndrome and those with clonal expansions of large granular lymphocytes in rheumatoid arthritis.

OBJECTIVE: Patients with chronic clonal proliferation of large granular lymphocytes (LGL leukemia) often have splenomegaly, neutropenia, and rheumatoid arthritis (RA), thereby resembling the manifestations observed in patients with Felty's syndrome. The present study sought to determine whether patients with these disorders represent 2 distinct subsets of neutropenic RA. METHODS: Prospective cohort study of outpatients attending clinics in university and private hospitals and in offices of private practice physicians. Twenty-two patients with Felty's syndrome and 22 patients with LGL leukemia, 10 of whom had RA, were studied. HLA genotyping was performed on peripheral blood mononuclear leukocyte genomic DNA. RESULTS: Nineteen of the 22 patients with Felty's syndrome (86%) were DR4 positive. Nine of the 10 patients with LGL leukemia plus RA were also DR4 positive. In contrast, only 4 of the 12 patients with LGL leukemia without RA (33%) were DR4 positive, a frequency that was within the normal range. CONCLUSION: The finding of an equally high prevalence of DR4 in patients with Felty's syndrome and in those with LGL leukemia plus RA suggests that both disorders have a similar immunogenetic basis and are parts of a single disease process rather than 2 separate disorders.

TAP2D is associated with HLA-B44 and DR4 and may contribute to rheumatoid arthritis and Felty's syndrome susceptibility.

OBJECTIVES: TAP2 transporter gene polymorphisms have been ascertained in patients with rheumatoid arthritis (RA) and Felty's syndrome (FS) to determine whether particular alleles of this gene are disease associated. METHODS: TAP2 dimorphisms at amino acid positions 379, 565 and 665 were detected using ARMS-PCR in 89 RA patients, 24 FS patients and 64 control subjects. TAP 2 alleles were assigned from these results. RESULTS: The frequency of one particular allele, TAP2D, was increased in both RA (OR 2.6, 95% CI 1.2 - 5.8) and FS (OR 3.9, 95% CI 1.4 - 10.7). When individual amino acid polymorphisms were compared between patients and controls, isoleucine at position 379 (present in TAP2D and TAP2C) was significantly increased, indicating that this dimorphism itself may be associated with RA (OR 5.0, 95% CI 2.4 - 10.2) and FS (OR 5.0, 95% CI 1.91 - 3.2). DISCUSSION: The presence of TAP2D was greatly increased in HLA-B44/DR4 positive RA (83%) and FS (67%) patients. These frequencies were appreciably higher than in the HLA-B44/DR4 controls (11%), suggesting that linkage disequilibrium alone may not explain the increase in TAP2D frequency in patients and that this allele may represent an additional risk factor in these conditions.

Tumor necrosis factor alpha-308 gene variants in relation to major histocompatibility complex alleles and Felty's syndrome.

The location of the human TNF genes within the MHC complex has prompted much speculation about the role of TNF alleles in the etiology of MHC-associated autoimmune diseases. On sequencing the 5' regulatory region of the human TNFA gene a G (TNFA-308G) to A (TNFA-308A) transition polymorphism at position -308 was discovered. We have developed a simple PCR assay to facilitate the screening of the -308 polymorphism at the DNA level. In view of the possible linkage between the TNFA-308A allele and a certain MHC type, TNFA-308 genotypes in HLA-typed healthy individuals (n = 88) were determined. A statistically significant association between the TNFA-308A allele and HLA-DR3, DQB1*0201, DQA1*0501, A1, B8, and the NcoI 5.5-kb RFLP of the TNFB gene was observed. In addition, we determined the frequency of the TNFA-308A allele in patients with FS (n = 13), an HLA-DR4-associated disease. In this study, no association was found of Felty's syndrome with the TNFA-308A allele, indicating that this allele does not appear to be a susceptibility factor for FS.

The large granular lymphocyte syndrome with rheumatoid arthritis. Immunogenetic evidence for a broader definition of Felty's syndrome.

OBJECTIVE: To assess whether the HLA-DR4 association found in rheumatoid arthritis (RA) is also seen in the large granular lymphocyte (LGL) syndrome. METHODS: HLA-DR genotyping was performed using restriction fragment length polymorphism and polymerase chain reaction analysis. RESULTS: LGL syndrome patients with RA showed the same HLA-DR4 association seen in RA/Felty's syndrome (FS), while LGL syndrome patients without arthritis did not. CONCLUSION: It is proposed that FS and the LGL syndrome represent different variants of a broader syndrome comprising RA, neutropenia, LGL expansions, HLA-DR4 positivity, and splenomegaly.

HLA-DPA1 and HLA-DPB1 in rheumatoid arthritis and its subsets.

The aim of this study was to examine the relationship between HLA-DP and susceptibility to articular and extra-articular features (Felty's syndrome and vasculitis) of rheumatoid arthritis (RA). The possible association of DP types with severity of articular disease was also analysed. No statistically significant associations were observed between HLA-DP alleles and articular or extra-articular features of RA, or to the severity of the arthritis when p was corrected for the number of alleles tested.

Analysis of lymphocyte phenotype and T cell receptor genotype in Felty's syndrome.

Natural killer (NK) cells and CD3+ large granular lymphocytes (LGL) were investigated in patients with Felty's syndrome (FS), rheumatoid arthritis (RA) and healthy controls. In most patients with FS, NK cell number and activity were decreased. CD3+ LGL were unchanged. However, in one patient a marked expansion of CD3- CD16+ CD56+ (NK) cells was seen and in a second, an expansion of CD3+ LGL. In FS there was also an increase in HLA- DR+ and CD8+ but not gamma delta+ T cells. Three of 11 patients with FS studied demonstrated a dominant rearrangement of the T cell receptor beta gene constant region consistent with oligoclonal T cell expansion.