Patricia Arscott

TRAIL Death Pathway Expression and Induction in Thyroid Follicular Cells

To determine whether programmed cell death in thyroid follicular cells can be related to activation of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway, we examined the expression and function of this pathway in primary thyroid follicular cells and a papillary thyroid carcinoma cell line in vitro. Despite the expression of TRAIL receptors death receptor 4 and death receptor 5, purified TRAIL could not induce programmed cell death (PCD) in any of the thyroid follicular cells examined. However, pre-incubation with cycloheximide before TRAIL facilitated the induction of rapid and massive PCD. This suggested that despite the presence of a labile inhibitor of the TRAIL pathway, TRAIL could mediate PCD under appropriate conditions. To determine whether there were sources of TRAIL in the thyroid that could interact with thyroid follicular cell TRAIL receptors, RNase protection assays were used to determine TRAIL mRNA expression. TRAIL message was expressed in intrathyroidal lymphocytes isolated from a patient with thyroiditis, and unexpectedly, thyroid follicular cells themselves could be induced to express abundant TRAIL message in the presence of the inflammatory cytokines interferon γ, tumor necrosis factor α, and interleukin 1β. Furthermore, the papillary thyroid carcinoma cell line could be induced to kill the TRAIL-sensitive lymphoma cell line BJAB through a TRAIL-dependent mechanism.

INFLAMMATORY CYTOKINE REGULATION OF FAS-MEDIATED APOPTOSIS IN THYROID FOLLICULAR CELLS

The occurrence of apoptosis in thyroid follicular cells induced by Fas activation has been a subject of much debate. This is due, in part, to the fact that no physiologically relevant treatment conditions have been reported to cause rapid and extensive Fas-mediated apoptosis in thyroid cells, whereas treatment with the protein synthesis inhibitor cycloheximide prior to Fas activation allows for massive cell death. This indicates that the Fas signaling pathway is present but that its function is blocked in the overwhelming majority of cultured thyroid cells. To reconcile the conflicting reports, we set out to identify physiologically relevant conditions in which rapid, massive thyroid cell apoptosis in response to Fas activation could be demonstrated. We determined that susceptibility to Fas-activated apoptosis could be influenced by certain combinations of inflammatory cytokines. Although no single cytokine was effective, pretreatment of thyroid cells with the combination of γ-interferon and either tumor necrosis factor-α or interleukin 1β allowed for massive Fas-mediated apoptosis. Susceptibility to Fas-induced death correlated with an increase in expression of a tunicamycin-inhibitable high molecular weight form of Fas but not with aggregate expression of Fas.

Fas (APO-1, CD95)-Mediated Apoptosis in Thyroid Cells Is Regulated by a Labile Protein Inhibitor.

To determine whether thyroid cell apoptosis observed in autoimmune thyroid disease could be related to activation of the Fas pathway, we examined the expression and function of Fas on thyroid follicular cells in vitro. Fas messenger RNA was found to be present using two different techniques and was expressed at equal levels in thyrocytes cultured either in the presence or absence of TSH. Fas antigen protein expression was demonstrated by Western blot of thyroid cell lysates and by immunohistochemical staining of thyrocytes, and the amount of Fas protein present did not appear to vary regardless of culture conditions. Despite expressing substantial amounts of Fas protein, thyrocytes treated with anti-Fas monoclonal antibody failed to undergo apoptosis. The addition of either interferon-gamma or interleukin-1beta to the anti-Fas-treated cell cultures also did not promote apoptotic signaling through this pathway. In contrast, the concomitant administration of cycloheximide allowed the induction of apoptosis through the activation of Fas in thyrocytes. These results suggest that Fas is constitutively expressed in thyrocytes, but that the induction of apoptosis through the Fas pathway is blocked by a labile protein inhibitor.

A Unique Combination of Inflammatory Cytokines Enhances Apoptosis of Thyroid Follicular Cells and Transforms Nondestructive to Destructive Thyroiditis in Experimental Autoimmune Thyroiditis

Treatment of cultured primary human thyroid cells with IFN-γ and TNF-α uniquely allows the induction of Fas-mediated apoptosis. To investigate the role of this cytokine combination in vivo, CBA/J mice were immunized with thyroglobulin and then injected with IFN-γ and TNF-α. Compared with control animals, mice treated with IFN-γ and TNF-α showed significantly sustained lymphocytic infiltration in the thyroid, which was associated with the destruction of portions of the follicular architecture at wk 6 after initial immunization. Furthermore, the number of apoptotic thyroid follicular cells was increased only in the thyroids from mice treated with the IFN-γ and TNF-α. We also analyzed the function of the Fas pathway in vivo in cytokine-treated mice by using an agonist anti-Fas Ab injected directly into the thyroid. Minimal apoptosis of thyroid epithelial cells was observed unless the mice were pretreated with IFN-γ and TNF-α. These data demonstrate that this unique combination of inflammatory cytokines facilitates the apoptotic destruction of thyroid follicular cells in experimental autoimmune thyroiditis, in a manner similar to what is observed in Hashimoto’s thyroiditis in humans.

2-Methoxyestradiol, an endogenous estrogen metabolite, induces thyroid cell apoptosis

The etiology of autoimmune thyroid diseases is unclear; however, the extreme female predominance suggests that sex hormones may have a pathogenic role. 2-Methoxyestradiol (2-ME) is present in the serum of women during the ovulatory and luteal phases of the menstrual cycle, and during pregnancy. We investigated the actions of 2-ME and estrogen on thyroid follicular cells. 2-ME induced dramatic changes in cell morphology and decreased the viability of the cells, as well as disrupted the structural integrity of cultured thyroid follicles. Flow cytometric analysis showed that 2-ME halted cell proliferation by arresting the cells in the G2/M cell-cycle compartment. Prolonged exposure to 2-ME led to apoptosis and to increased release of the autoantigen thyroid peroxidase (TPO). 17beta-estradiol failed to produce a similar effect even in 40-fold molar excess to 2-ME. Co-treatment with estrogen receptor antagonists did not alter the 2-ME effect, indicating that 2-ME was not operating through a classic nuclear estrogen receptor. In conclusion, this study indicates that 2-ME induces G2/M cycle arrest, apoptosis and the disruption of thyroid follicles. This process results in the release of thyroid antigens that may play a role in high incidence of thyroid autoantibodies and autoimmune thyroid disease in women.

Low yield of genetic testing for known vascular connective tissue disorders in patients with fibromuscular dysplasia.

Patients with fibromuscular dysplasia (FMD) may have clinical features consistent with Mendelian vascular connective tissue disorders. The yield of genetic testing for these disorders among patients with FMD has not been determined. A total of 216 consecutive patients with FMD were identified. Clinical characteristics were collected and genetic test results reviewed for abnormalities in the following genes: transforming growth factor-β receptor 1 and 2 (TGFβR1 and TGFβR2), collagen 3A1, fibrillin-1, smooth muscle α-actin 2, and SMAD3. A total of 63 patients (63/216; 29.2%) were referred for genetic counseling with testing performed in 35 (35/63; 55.6%). The percentage of patients with a history of arterial or aortic dissection, history of aortic aneurysm, systemic features of a connective tissue disorder, and a family history of sudden death was significantly larger in the group that underwent genetic testing (62.9% vs 18.2%, p < 0.001; 8.6% vs 1.7%, p = 0.02; 51.4% vs 17.1%, p < 0.001; and 42.9% vs 22.7%, p = 0.04, respectively). Two patients were found to have distinct variants in the TGFβR1 gene (c.611 C>T, p.Thr204lle and c.1285 T>C, p.Tyr429His). The yield of genetic testing for vascular connective tissue disorders was low in a high-risk subset of FMD patients. However, two patients with a similar phenotype had novel and distinct variants in the TGFβR1 gene, a finding which merits further investigation.

Care in Specialized Centers and Data Sharing Increase Agreement in Hypertrophic Cardiomyopathy Genetic Test InterpretationCLINICAL PERSPECTIVE

Background— Clinically impactful differences in the interpretation of genetic test results occur between laboratories and clinicians. To improve the classification of variants, a better understanding of why discrepancies occur and how they can be reduced is needed. Methods and Results— We examined the frequency, causes, and resolution of discordant variant classifications in the Sarcomeric Human Cardiomyopathy Registry (SHaRe), a consortium of international centers with expertise in the clinical management and genetic architecture of hypertrophic cardiomyopathy. Of the 112 variants present in patients at >1 center, 23 had discordant classifications among centers (20.5%; Fleiss &kgr;, 0.54). Discordance was more than twice as frequent among clinical laboratories in ClinVar, a public archive of variant classifications (315/695 variants; 45.2%; Fleiss &kgr;, 0.30; P<0.001). Discordance in SHaRe most frequently occurred because hypertrophic cardiomyopathy centers had access to different privately held data when making their classifications (75.0%). Centers reassessed their classifications based on a comprehensive and current data summary, leading to reclassifications that reduced the discordance rate from 20.5% to 10.7%. Different interpretations of rarity and co-occurrence with pathogenic variants contributed to residual discordance. Conclusions— Discordance in variant classification among hypertrophic cardiomyopathy centers is largely attributable to privately held data. Some discrepancies are caused by differences in expert assessment of conflicting data. Discordance was markedly lower among centers specialized in hypertrophic cardiomyopathy than among clinical laboratories, suggesting that optimal genetic test interpretation occurs in the context of clinical care delivered by specialized centers with both clinical and genetics expertise.

Genetic testing impacts the utility of prospective familial screening in hypertrophic cardiomyopathy through identification of a nonfamilial subgroup

PurposeHypertrophic cardiomyopathy (HCM) is considered a hereditary autosomal dominant condition, but genetic testing is positive in only half of patients. In patients with negative genetic tests, the inheritance pattern and utility of family screening are unclear.MethodsSubjects with HCM were prospectively enrolled in a registry. A survey at a median follow-up of 4 years determined the yield of family screening.ResultsThe outcome of cardiac screening on 267 family members was reported by 120 survey respondents. Subjects with positive genetic test or family history (n=74, 62%) reported an HCM diagnosis in 34 of 203 first-degree relatives who were screened (17%). Affected family members were diagnosed at a mean age of 30–39 years, and 22 of 34 experienced HCM-related adverse events (65%). Gene test–negative subjects with no prior family history of HCM (n=46, 38%) reported an HCM diagnosis in only 2 of 64 first-degree relatives who were screened (3%, p<0.001). These two individuals were diagnosed at age >40 years without HCM-related adverse events.ConclusionHypertrophic cardiomyopathy is a heterogeneous disorder, only half of which tracks with a Mendelian inheritance pattern. Negative genetic testing and family history indicates a more complex genetic basis corresponding to low risk for family members.

A Case for Inclusion of Genetic Counselors in Cardiac Care.

Recent advances in genetic testing for heritable cardiac diseases have led to an increasing involvement of the genetic counselor in cardiology practice. We present a series of cases collected from a nationwide query of genetics professionals regarding issues related to cost and utilization of genetic testing. Three themes emerged across cases: (1) choosing the most appropriate genetic test, (2) choosing the best person to test, and (3) interpreting results accurately. These cases demonstrate that involvement of a genetic counselor throughout the evaluation, diagnosis, and continuing management of individuals and families with inherited cardiovascular conditions helps to promote the efficient use of healthcare dollars.

Apoptosis and the thyroid: The biology and potential implications for thyroid disease

Apoptosis is a physiologic process of cell death that is central to normal development and occurs in response to a variety of physiologic and pathophysiologic stimuli. In the thyroid, abnormal apoptotic activity may be involved in a variety of diseases such as Hashimoto thyroiditis and Graves disease. Receptor-mediated apoptosis signaling pathways such as those initiated by Fas ligand, tumor necrosis factor, and tumor necrosis factor–related apoptosis-inducing ligand are present and functional in the thyroid. In Hashimoto thyroiditis, which is a typical organ-specific autoimmune disease resulting in thyroid destruction, Fas-mediated apoptosis has been suggested as a mechanism of thyroid follicular cell death. Graves disease is characterized by hyperthyroidism that appears to be caused by autoimmunity. This disorder may be linked to suppression of apoptosis. Apoptosis in thyroid follicular cells may be regulated at multiple levels, including the expression of anti-apoptotic proteins such as Bcl-2. Alterations in the expression of death receptors and ligands by inflammatory cytokines may also play a role in the regulation of apoptosis. These mechanisms may contribute to the pathogenesis of autoimmune thyroid disease.