Roberta Ricciardi

A genome-wide association study of myasthenia gravis

IMPORTANCE Myasthenia gravis is a chronic, autoimmune, neuromuscular disease characterized by fluctuating weakness of voluntary muscle groups. Although genetic factors are known to play a role in this neuroimmunological condition, the genetic etiology underlying myasthenia gravis is not well understood. OBJECTIVE To identify genetic variants that alter susceptibility to myasthenia gravis, we performed a genome-wide association study. DESIGN, SETTING, AND PARTICIPANTS DNA was obtained from 1032 white individuals from North America diagnosed as having acetylcholine receptor antibody-positive myasthenia gravis and 1998 race/ethnicity-matched control individuals from January 2010 to January 2011. These samples were genotyped on Illumina OmniExpress single-nucleotide polymorphism arrays. An independent cohort of 423 Italian cases and 467 Italian control individuals were used for replication. MAIN OUTCOMES AND MEASURES We calculated P values for association between 8,114,394 genotyped and imputed variants across the genome and risk for developing myasthenia gravis using logistic regression modeling. A threshold P value of 5.0×10(-8) was set for genome-wide significance after Bonferroni correction for multiple testing. RESULTS In the overall case-control cohort, we identified association signals at CTLA4 (rs231770; P=3.98×10(-8); odds ratio, 1.37; 95% CI, 1.25-1.49), HLA-DQA1 (rs9271871; P=1.08×10(-8); odds ratio, 2.31; 95% CI, 2.02-2.60), and TNFRSF11A (rs4263037; P=1.60×10(-9); odds ratio, 1.41; 95% CI, 1.29-1.53). These findings replicated for CTLA4 and HLA-DQA1 in an independent cohort of Italian cases and control individuals. Further analysis revealed distinct, but overlapping, disease-associated loci for early- and late-onset forms of myasthenia gravis. In the late-onset cases, we identified 2 association peaks: one was located in TNFRSF11A (rs4263037; P=1.32×10(-12); odds ratio, 1.56; 95% CI, 1.44-1.68) and the other was detected in the major histocompatibility complex on chromosome 6p21 (HLA-DQA1; rs9271871; P=7.02×10(-18); odds ratio, 4.27; 95% CI, 3.92-4.62). Association within the major histocompatibility complex region was also observed in early-onset cases (HLA-DQA1; rs601006; P=2.52×10(-11); odds ratio, 4.0; 95% CI, 3.57-4.43), although the set of single-nucleotide polymorphisms was different from that implicated among late-onset cases. CONCLUSIONS AND RELEVANCE Our genetic data provide insights into aberrant cellular mechanisms responsible for this prototypical autoimmune disorder. They also suggest that clinical trials of immunomodulatory drugs related to CTLA4 and that are already Food and Drug Administration approved as therapies for other autoimmune diseases could be considered for patients with refractory disease.

Management of pleural recurrence after curative resection of thymoma

OBJECTIVE A complete surgical resection is the cornerstone of therapy of thymic tumors. Unfortunately, there is no standard treatment for pleural recurrence. This article describes our overall experience with the surgical treatment of pleural implants in patients who previously underwent resection of a thymoma. MATERIAL AND METHODS From January 1980 to June 2006, 20 patients previously operated on for a thymoma were operated on for the surgical resection of pleural implants. Patients with the initial Masaoka stage IVA were excluded from our analysis. Our sample comprised 10 male and 10 female patients (12-65 years old). The surgical approach to the resection of the thymoma was as follows: video-assissted thoracic surgery in 2 patients, sternotomy in 13 patients, thoracotomy in 2 patients, and sternothoracotomy in 3 patients. The initial Masaoka stage of the thymoma was IIA in 2 patients, IIB in 7 patients, and III in 11 patients. RESULTS The interval between resection of the thymoma and pleural implants ranged from 11 to 156 (median 60) months. Fifteen patients had a thymus-related syndrome (in 13 patients it resulted myasthenia gravis), and in 11 patients it improved or remitted after treatment of the pleural recurrence. All the resections were performed through a posterolateral thoracotomy. Three patients underwent an iterative resection of new pleural implants. At the latest follow-up, 10 patients are still alive (8 disease-free) and 10 have died (9 of a relapse and 1 of the complications of red cell aplasia). From the pleural recurrence resection, the overall 5- and 10-year survivals are 43.1% and 25.8%, respectively. CONCLUSIONS Repeat operation on patients with thymoma pleural recurrences is feasible and safe. It can produce satisfactory results in terms of overall survival and paraneoplastic syndrome control. Moreover, the multimodality treatment could improve the results of surgical treatment.

Association of thymoma and myasthenia gravis: oncological and neurological results of the surgical treatment

BACKGROUND Thymoma occurs in about 10-20% of myasthenic patients and in turn, 20-25% of patients with a thymoma have myasthenia gravis. Both diseases are treated by surgery. The aims of this study were to analyze the clinical features of these patients and the oncological and neurological outcomes after thymectomy. METHODS Clinical and pathological data, complete remission rate as well as overall survival rates were retrospectively analyzed in a cohort of myasthenic patients who underwent extended thymectomy for thymoma between 1993 and 2006. RESULTS One hundred and twenty-three patients (60 m and 63 f) with a mean age of 56 years (range 22-83) underwent extended thymectomy. The WHO histological classification was: A in 22 cases, AB in 18, B1 in 33, B2 in 22, and B3 in 28. The Masaoka clinical staging was: I in 10 cases, IIA in 33, IIB in 50, III in 14, IVA in 15, IVB in 1. We experienced 2 postoperative deaths. With a overall mean follow-up of 76 months 42 patients had a complete remission, 39 a remission with medications, 35 an improvement of the symptoms, 3 remained nearly in the same status and 4 worsened. At the last follow-up, 112 patients were alive; 11 with disease. Four deaths were related to the tumor. Actuarial 5- and 10-year survival was 93.4% and 79.6%, respectively. CONCLUSIONS Neurological outcome of the extended thymectomy in myasthenic thymoma patients was satisfactory. Higher complete remission rate is expected in early stage thymoma. Regarding the overall survival it was dependent on the Masaoka stage, the WHO classification and the achievement of complete remission of myasthenic symptoms.

Extended thymectomy in myasthenia gravis: a team-work of neurologist, thoracic surgeon and anaesthesist may improve the outcome

OBJECTIVE We reviewed our overall experience on 163 patients, affected by myasthenia gravis, who underwent thymectomy between 1976 and 1998. A comparison between the oldest series of 72 patients (January 1976-December 1992), referred by various neurologists and operated on through different approaches, and the last 91 patients (January 1993-December 1998), taking part in a strict diagnostic-therapeutical programme, was made. METHODS Anagraphic data, duration of symptoms, the surgical approach, necessity of respiratory assistance, the hospital stay, histopathological findings, preoperative and postoperative Osserman classification, as well as medications, were globally analyzed and then compared in the two groups. RESULTS Significant differences in the length of hospitalization (8.7 days vs. 4.2 days; P=0.00001) and in the prolonged intubation rate (18 vs. 0; P<0.000001) were observed in the most recent series. Patients in the pre-operative Osserman stage I and operated on in the second period had a higher complete remission rate at the univariate analysis (P<0.001 and P<0.0001, respectively). At the multivariate analysis the only parameter which affected the outcome was to be operated on in the second period (P<0.01). CONCLUSIONS Our experience confirms the role of the extended thymectomy in the treatment of myasthenia gravis. Whenever an extended thymectomy was performed through a complete sternotomy it was a quick procedure, with short hospitalization and acceptable cosmetic results. A careful pharmacological control of the myasthenic symptoms and the presence of team-work among neurologist, thoracic surgeon and anaesthesist in the peri-operative setting reduce the incidence of complications and might increase the efficacy of the thymectomy.

Change in myasthenia gravis epidemiology in Trento, Italy, after twenty years.

Background: The recent literature suggests that the incidence and prevalence of myasthenia gravis (MG) are changing. We performed a population-based study of MG in the province of Trentino (524,826 inhabitants) and compared the results with those collected 20 years ago. Methods: Multiple sources of information (discharge diagnosis, antibody tests and anticholinesterase drugs) were analyzed. Incidence was calculated from 2005 to 2009. Prevalence was calculated on December 31, 2009. Comparison was made with descriptive statistics for 1981–1990 for the identical region. Results: Incidence and prevalence greatly increased in comparison with 1981–1990 data. The prevalence rate increased from 82.9 (95% confidence interval, CI, 58.4–114.3) in 1990 to 129.6 (95% CI 100.6–164.3) per million population, whereas the average annual incidence increased from 7.4 (95% CI 5–10.4) per million person-years in 1981–1990 to 14.8 (95% CI 10.5–20.3) in 2005–2009. This increase was mainly due to male patients with late-onset MG. Conclusions: The study confirms the increase in incidence and prevalence of late-onset MG in the same region 20 years apart. So we should consider MG also as a disease of the elderly.

PTPN22 and myasthenia gravis: replication in an Italian population and meta-analysis of literature data.

Polymorphisms in PTPN22 are associated with many autoimmune diseases; while rs2476601 is supposed to play a major role, other experimental data suggest that rs2488457 may be even more important. Results in myasthenia gravis are controversial. In 356 Italian myasthenic patients and 439 controls genotyped for both polymorphisms, we found that rs2476601 was not associated with myasthenia, presence of autoantibodies, thymus pathology, sex or onset age unlike previous studies on other European populations (confirmed by the present meta-analysis). On the other hand, while rs2488457 was not associated with myasthenia or thymus pathology, we found a correlation of rs2488457 with low autoantibody titers and a trend of association with a less severe disease. Both polymorphisms were in tight linkage disequilibrium in controls, not in patients. Our results suggest that SNPs in this gene different from rs2476601, and/or epigenetic interactions, could play a greater role.

Association of the DNMT3B -579G>T Polymorphism with Risk of Thymomas in Patients with Myasthenia Gravis

Increasing evidence suggests a contribution of epigenetic processes in promoting cancer and autoimmunity. Myasthenia gravis (MG) is an autoimmune disease mediated, in approximately 80% of the patients, by antibodies against the nicotinic acetylcholine receptor (AChR+). Moreover, epithelial tumours (thymomas) are present in about 10-20% of the patients, and there is indication that changes in DNA methylation might contribute to the risk and progression of thymomas. However, the role of epigenetics in MG is still not completely clarified. In the present study we investigated if a common polymorphism (-579G>T: rs1569686) in the promoter of the DNMT3B gene coding for the DNA methyltransferase 3B, an enzyme that mediates DNA methylation, increases the risk to develop MG or MG-associated thymomas. The study polymorphism was selected based on recent reports and a literature meta-analysis suggesting association with increased risk of various types of cancer. We screened 324 AChR+ MG patients (140 males and 184 females, mean age 56.0 ± 16.5 years) and 735 healthy matched controls (294 males and 441 females, mean age 57.3 ± 15.6 years). 94 of the total MG patients had a thymoma. While there was no association with the whole cohort of MG patients, we found a statistically significant association of the DNMT3B -579T allele (OR = 1.51; 95% CI=1.1-2.1, P = 0.01) and the TT homozygous genotype (OR = 2.59; 95% CI=1.4-4.9, P = 0.006) with the risk of thymoma. No association was observed in MG patients without thymoma, even after stratification into clinical subtypes. Present results suggest that the DNMT3B -579T allele might contribute to the risk of developing thymoma in MG patients, particularly in homozygous TT subjects.

IgG-specific cell-based assay detects potentially pathogenic MuSK-Abs in seronegative MG.

Objective: To increase the detection of MuSK-Abs using a CBA and test their pathogenicity. Methods: Sera from 69 MuSK-RIA–positive patients with myasthenia gravis (MG) (Definite MuSK-MG), 169 patients negative for MuSK-RIA and AChR-RIA (seronegative MG, SNMG), 35 healthy individuals (healthy controls, HCs), and 16 NMDA receptor-Ab–positive (NMDAR-Ab) disease controls were tested for binding to MuSK on a CBA using different secondary antibodies. Results: Initially, in addition to 18% of SNMG sera, 11% of HC and 19% of NMDAR-Ab sera showed positive binding to MuSK-transfected cells; this low specificity was due to anti-IgG(H+L) detection of IgM bound nonspecifically to MuSK. Using an IgG Fc gamma-specific secondary antibody, MuSK-Abs were detected by CBA in 68/69 (99%) of Definite MuSK-MG, 0/35 HCs, 0/16 NMDAR-Ab, and 14/169 (8%) of SNMG sera, providing increased sensitivity with high specificity. The RIA-negative, CBA-positive MuSK-IgG sera, but not IgM-MuSK–binding sera, reduced agrin-induced AChR clustering in C2C12 myotubes, qualitatively similar to RIA-positive MuSK-Abs. Conclusions: An IgG-specific MuSK-CBA can reliably detect IgG MuSK-Abs and increase sensitivity. In the MuSK-CBA, IgG specificity is essential. The positive sera demonstrated pathogenic potential in the in vitro AChR-clustering assay, although less effective than Definite MuSK-MG sera, and the patients had less severe clinical disease. Use of IgG-specific secondary antibodies may improve the results of other antibody tests. Classification of evidence: This study provides Class III evidence that an IgG-specific MuSK-CBA identifies patients with MG.

T-cell tumor necrosis factor-α receptor binding in myasthenic patients

Myasthenia gravis (MG) is a T-cell-dependent and antibody-mediated autoimmune disease of the neuromuscular junction, in which the cytokine network may be deranged. Specific receptors for tumor necrosis factor (TNF)-alpha, a cytokine with several effects on the neuroimmune system, were found on human lymphocytes. In the present study, we assayed TNF-alpha binding on peripheral blood T-cells from MG patients, finding that T-cells from patients have significantly more TNF-alpha receptors than those from controls (Bmax: 654 +/- 12 vs. 133 +/- 4 (mean +/- SEM) receptors/cell). Such TNF-alpha binding sites are of the same type in patients and healthy subjects (Kd: 68.7 +/- 4.3 vs. 70.1 +/- 4.8 (mean +/- SEM) pM). The enhanced T-cell TNF-alpha binding is due to an increased number of TNF-alpha receptors on T-helper lymphocytes. These results are discussed in terms of MG immunopathogenesis, since it has been reported that activated T-cells have increased amounts of TNF-alpha receptors.

T-cell interleukin-6 receptor binding in patients with myasthenia gravis

Myasthenia gravis (MG) is a T-cell-dependent and antibody-mediated autoimmune disease of the neuromuscular junction, in which the cytokine network may be deranged. Specific receptors for interleukin (IL)-6, a cytokine with several effects on the neuroimmune system, have been found on human lymphocytes. The aim of the present study has been to assay IL-6 binding on peripheral blood T cells from MG patients. We found that T cells from MG patients have significantly more IL-6 receptors than those from controls (Bmax: 334 +/- 6 vs 251 +/- 4 (mean +/- SEM) receptors/cell). Such IL-6 binding sites are of the same type in patients and healthy subjects (Kd: 26.5 +/- 0.7 vs 25.7 +/- 0.9 (mean +/- SEM) pM). The enhanced T-cell interleukin-6 binding is due to an increased number of interleukin-6 receptors on T-helper lymphocytes. These results are discussed in terms of MG immunopathogenesis, since it has been reported that activated T cells have increased amounts of IL-6 receptors.