Analysis of skewed X-chromosome inactivation in females with rheumatoid arthritis and autoimmune thyroid diseases.

INTRODUCTION: The majority of autoimmune diseases such as rheumatoid arthritis (RA) and autoimmune thyroid diseases (AITDs) are characterized by a striking female predominance superimposed on a predisposing genetic background. The role of extremely skewed X-chromosome inactivation (XCI) has been questioned in the pathogenesis of several autoimmune diseases. METHODS: We examined XCI profiles of females affected with RA (n = 106), AITDs (n = 145) and age-matched healthy women (n = 257). XCI analysis was performed by enzymatic digestion of DNA with a methylation sensitive enzyme (HpaII) followed by PCR of a polymorphic CAG repeat in the androgen receptor (AR) gene. The XCI pattern was classified as skewed when 80% or more of the cells preferentially inactivated the same X-chromosome. RESULTS: Skewed XCI was observed in 26 of the 76 informative RA patients (34.2%), 26 of the 100 informative AITDs patients (26%), and 19 of the 170 informative controls (11.2%) (P < 0.0001; P = 0.0015, respectively). More importantly, extremely skewed XCI, defined as > 90% inactivation of one allele, was present in 17 RA patients (22.4%), 14 AITDs patients (14.0%), and in only seven controls (4.1%, P < 0.0001; P = 0.0034, respectively). Stratifying RA patients according to laboratory profiles (rheumatoid factor and anti-citrullinated protein antibodies), clinical manifestations (erosive disease and nodules) and the presence of others autoimmune diseases did not reveal any statistical significance (P > 0.05). CONCLUSIONS: These results suggest a possible role for XCI mosaicism in the pathogenesis of RA and AITDs and may in part explain the female preponderance of these diseases.

Genetic polymorphisms in the EGFR (R521K) and estrogen receptor (T594T) genes, EGFR and ErbB-2 protein expression, and breast cancer risk in Tunisia.

We evaluated the association of epidermal growth factor receptor (EGFR) 142285G>A (R521K) and estrogen receptor alpha (ESR1) 2014G>A (T594T) single nucleotide polymorphisms with breast cancer risk and prognosis in Tunisian patients. EGFR 142285G>A and ESR1 2014G>A were genotyped in a sample of 148 Tunisian breast cancer patients and 303 controls using PCR-RFLP method. Immunohistochemitsry was used to evaluate the expression levels of EGFR, HER2, ESR1, progesterone receptor and BCL2 in tumors. We found no evidence for an association between EGFR R521K polymorphism and breast cancer risk. However, we found that the homozygous GG (Arg) genotype was more prevalent in patients with lymph node metastasis (P = .03) and high grade tumors (P = .011). The ESR1 2014G allele showed significant association with breast cancer risk (P = .025). The GG genotype was associated with HER2 overexpression and this association withstood univariate and multivariate analyses (P = .009; P = .021, resp.). These data suggest that the R521K might be a prognostic factor, because it correlates with both tumor grade and nodule status. The higher expression of HER2 in ESR1 T594T GG patients suggests the possibility that ESR1 gene polymorphisms accompanied by HER2 expression might influence the pathogenesis of breast cancers.

Aberrant BRAF splicing as an alternative mechanism for oncogenic B-Raf activation in thyroid carcinoma.

Activating BRAF mutations have recently been reported in 28-83% of papillary thyroid carcinomas (PTCs). However, it is not known whether aberrant BRAF splicing occurs in thyroid carcinoma. To investigate aberrant BRAF splicing and its association with BRAF mutation in thyroid tumours, we studied aberrant BRAF splicing and BRAF mutation from 68 thyroid tumours. BRAF(V600E) mutation was detected in 20 of 43 PTCs and all three anaplastic thyroid carcinomas (ATCs). There is a higher frequency of BRAF mutation in PTC patients with stage III and IV tumours compared with stage I and II. Novel BRAF splicing variants were detected in 12 PTCs, three follicular variants of PTC (FVPTCs), and one ATC, as well as in two thyroid carcinoma cell lines, ARO and NPA. These variants did not have the N-terminal auto-inhibitory domain of wild-type B-Raf, resulting in an in-frame truncated protein that contained only the C-terminal kinase domain and caused constitutive activation of B-Raf. These variants were significantly associated with advanced disease stage and BRAF(V600E) mutation (p < 0.001, Fisher exact test). Furthermore, expression of these variants in NIH3T3 and CHO cells could activate the MAP kinase signalling pathway, transform them in vitro, and induce tumours in nude mice. These data suggest that BRAF splicing variants may function as an alternative mechanism for oncogenic B-Raf activation. Combination of the BRAF(V600E) mutation and its splicing variants may contribute towards disease progression to poorly differentiated thyroid carcinoma.

Oncogenic activation of MAP kinase by BRAF pseudogene in thyroid tumors.

Activating BRAF mutations have been reported in 40% of papillary thyroid carcinomas (PTCs). The involvement of BRAF pseudogene in thyroid tumorigenesis has not previously been studied. We investigated BRAF pseudogene expression in 68 thyroid tumors: 16 multinodular goiters, 43 classic PTCs, 6 follicular variants of PTC, and 3 anaplastic thyroid carcinomas. BRAF pseudogene function was studied by Western blots, soft agar assay, and tumorigenesis in nude mice. BRAF pseudogene expression was detected in 7 multinodular goiters, 18 classic PTC, and 1 follicular variants of PTC. There is an inverse correlation between BRAF pseudogene expression and BRAF mutation. The pseudogene transcripts were more frequently detected in tumors without BRAF mutation than those with BRAF mutation. Furthermore, BRAF pseudogene expression could activate the MAP kinase signaling pathway, transform NIH3T3 cells in vitro, and induce tumors in nude mice. These data suggest that BRAF pseudogene activation may play a role in thyroid tumor development.

Lack of association of VDR gene polymorphisms with thyroid autoimmune disorders: familial and case/control studies.

We study the association between three Vitamin D receptor gene polymorphisms (rs10735810, rs1544410, rs731236) and susceptibility to thyroid autoimmune diseases. Seventy-six affected subjects, belonging to a large family, as well as one hundred unrelated Tunisian patients and one hundred healthy Tunisian controls were genotyped. A family-based association test and a standard chi-square test were used to assess association in family and case-control data, respectively. Our results showed no significant association of the Vitamin D receptor gene polymorphisms with the susceptibility to thyroid autoimmune diseases in the family. Moreover, allele frequencies for the three polymorphisms in the Tunisian population were similar to those reported in the Tunisian control population and none was associated with the disease. These results suggest a lack of association between the Vitamin D receptor gene polymorphisms and susceptibility to thyroid autoimmune diseases in Tunisian population, in agreement with data from the UK, but in conflict with studies from the Far East.

Cleavage of the human thyrotropin receptor by ADAM10 is regulated by thyrotropin.

The thyrotropin receptor (TSHR) has a unique 50 residue (317-366) ectodomain insertion that sets it apart from other glycoprotein hormone receptors (GPHRs). Other ancient members of the leucine-rich repeat G protein-coupled receptor (GPCR) (LGR) family do exhibit ectodomain insertions of variable lengths and sequences. The TSHR-specific insert is digested, apparently spontaneously, to release the ectodomain (A-subunit) leaving the balance of the ectodomain attached to the serpentine (B-subunit). Despite concerted efforts for the last 12 years by many laboratories, the enzyme involved in TSHR cleavage has not been identified and a physiologic role for this process remains unclear. Several lines of evidence had suggested that the TSHR protease is likely a member of the a disintegrin and metalloprotease (ADAM) family of metalloproteases. We show here that the expression of ADAM10 was specific to the thyroid by specially designed DNA microarrays. We also show that TSH increases TSHR cleavage in a dose-dependent manner. To prove that ADAM10 is indeed the TSHR cleavage enzyme, we investigated the effect of TSH-induced cleavage by a peptide based on a motif (TSHR residues 334-349), shared with known ADAM10 substrates. TSH increased dose dependently TSHR ectodomain cleavage in the presence of wild-type peptide but not a scrambled control peptide. Interestingly, TSH increased the abundance of non-cleaved single chain receptor, as well higher molecular forms of the A-subunit, despite their enhancement of the appearance of the fully digested A-subunit. This TSH-related increase in TSHR digested forms was further increased by wild-type peptide. We have identified for the first time ADAM10 as the TSHR cleavage enzyme and shown that TSH regulates its activation.

Ribonucleic acid interference targeting S100A4 (Mts1) suppresses tumor growth and metastasis of anaplastic thyroid carcinoma in a mouse model.

CONTEXT: The characteristic feature of malignant neoplasm is invasion and metastasis. Despite advances in the management of thyroid carcinoma and other solid tumors, metastasis continues to be the most significant cause in cancer mortality. OBJECTIVE: Our objective was to examine the effects of S100A4 expression knockdown by RNA interference on the growth and metastasis of human anaplastic thyroid carcinoma cells (ARO) and the sensibility of ARO to paclitaxel after S100A4 knockdown. DESIGN: A plasmid construct was made that expressed small hairpin RNA (shRNA) specific for S100A4. The construct was stably transfected into ARO cells (ARO/S100A4-shRNA). The tumorigenicity, metastatic potential, and sensibility of ARO/S100A4-shRNA to paclitaxel were investigated. RESULTS: S100A4 expression was reduced by 71.3 +/- 4.7% in ARO/S100A4-shRNA by real-time RT-PCR analysis. The growth rate of ARO/S100A4-shRNA was reduced by 46 +/- 7.6% in a cell proliferation assay. Cell cycle analysis showed increased G(2)/M accumulation in ARO/S100A4-shRNA. Tumor formation and growth induced by sc injection of 5 x 10(6) ARO/S100A4-shRNA into the nude mice were significantly reduced, and no tumor metastasis was found in any of the mice. We also demonstrated significant induction of apoptosis in ARO/S100A4-shRNA after incubation with 15 nm paclitaxel, indicating that tumor cells were sensitized to chemotherapy as a result of S100A4 knockdown. CONCLUSIONS: These data suggest that reduction of S100A4 by RNA interference is a viable approach to inhibit tumor growth and metastasis. Given that S100A4 is overexpressed in many kinds of tumors, the current study provides the proof of concept in its therapeutic potential.

Different genotype distribution of the GNB3 C825T polymorphism of the G protein beta3 subunit in adenomas and differentiated thyroid carcinomas of follicular cell origin.

A C825T polymorphism has been demonstrated in the GNB3 gene that encodes the Gbeta3 subunit of heterotrimeric G proteins. Due to enhanced G protein activation, the GNB3 825T allele is associated with an increased signal transduction activity. To elucidate a possible role in the development and course of thyroid tumours of follicular cell origin, C825T polymorphism genotypes and allele frequencies were investigated in a series of adenomas and differentiated carcinomas. Genotypes and the allele frequency of the Gbeta3 polymorphism were investigated in samples from 361 patients (all white Caucasians) with differentiated thyroid tumours of follicular cell origin [80 adenomas and 95 follicular (FTCs) and 186 papillary carcinomas (PTCs)]. The results were compared with those of 1859 healthy controls. Both the genotype distribution (p = 0.029) and the allele frequency (p = 0.028) of the adenoma group were statistically significantly different from those of the control group. Thyroid adenomas also differed for both parameters significantly from FTCs (p = 0.042 and 0.033, respectively) and PTCs (0.0018 and 0.0081, respectively), whereas no statistical difference was noted between the FTC and PTC groups. Although the biological significance of these observations remains obscure, the results are suggestive of a putative role for the GNB3 polymorphism in thyroid tumour development and/or progression. Further research has to elucidate if, and to what extent, this common germ-line variation influences the TSH-triggered signalling pathways responsible for thyroid function and proliferation.

Well differentiated thyroid carcinoma is associated with human lymphocyte antigen D-related 11 in Eastern Hungarians: a case of changing circumstances.

BACKGROUND: Using serologic human lymphocyte antigen (HLA) typing, the authors previously described a strong association between well differentiated thyroid carcinoma and HLA D-related 1 (HLA-DR1) in a population of unselected patients from Eastern Hungary. METHODS: In the current study, the authors used polymerase chain reaction-single strand conformational polymorphism to determine the HLA-DR type in 75 patients with well differentiated thyroid carcinoma from the same area as their previous population, and they compared the current results with the results from a group of 170 healthy controls. RESULTS: A significant increase in HLA-DR11, rather than HLA-DR1, was observed in patients with well differentiated thyroid carcinoma among a population of patients from the same area that was studied previously. After excluding technical reasons to account for differences in disease association, they postulated that interim environmental factors, possibly radiation fall-out, may have resulted in differences in genetic susceptibility to thyroid carcinoma. Consideration of the potential antigenic peptides that may be restricted by the two HLA-DR alleles may have allowed for the binding of similar peptides to initiate an immune response, likely leading to progressive immunomodulation of the tumor. Discriminat function analysis indicated a significant relation between tumor size and metastases and less lymphocytic infiltration of the tumor, but this was not related to HLA-DR phenotypes. CONCLUSIONS: The authors found that the study of major histocompatability complex alleles holds promise for understanding the events that initiate and maintain tumor immunomodulation.

Microarray analysis of metastasis-associated gene expression profiling in a murine model of thyroid carcinoma pulmonary metastasis: identification of S100A4 (Mts1) gene overexpression as a poor prognostic marker for thyroid carcinoma.

Tumor cell invasion and metastasis are the hallmark of malignant neoplasm. Despite advances in the management of thyroid carcinoma and other solid tumors, metastasis continues to be the most significant cause in cancer mortality. To gain new insights into this complex process in thyroid carcinoma, we established a thyroid carcinoma cell line (ARO-met2) with high metastatic capacity to the lung by sequential passage of a human anaplastic thyroid cancer cell line (ARO) through the lung of a nude mouse. Global patterns of gene expression were analyzed in cells of the parental ARO and the ARO-met2, using Atlas human cancer 1.2 array with 1176 cancer-related genes. In total, 184 genes were differentially expressed more than 1.5 times, and 64 genes were differentially expressed over two times. Among those 64 genes, 43 were overexpressed, and 21 genes were underexpressed. Many genes whose increased expression was thought to be related to tumor progression were identified, such as c-Met, ezrin, integrin, motility-related protein-1, cadherin, and S100A4. The most highly expressed gene is the S100A4 (8-fold higher than control), which is a member of a small calcium binding protein family and is involved in the cell proliferation and cancer progression. The S100A4 overexpression in the ARO-met2 cells was later confirmed by Northern blot and real-time reverse transcriptase-PCR. Analysis of 49 thyroid tumor specimens by real-time reverse transcriptase-PCR (eight benign goiters, 36 papillary, and five anaplastic carcinomas) revealed that S100A4 overexpression was present in most advanced thyroid carcinomas and lymph node metastases, and was associated with poor prognosis. None of the benign goiters was found to have S100A4 overexpression. These data suggest that S100A4 could be used as a prognostic marker for thyroid carcinoma. Given that S100A4 is involved in tumor progression and metastasis, it may be a potential target for therapeutic intervention.

Minireview: structural and functional evolution of the thyrotropin receptor.

TSH receptor (TSHR) is a member of the leucine-rich repeat-containing G protein-coupled receptors. Both TSHR and its ligand TSH have evolved to acquire specificity, minimize cross-reaction to other glycoprotein hormone receptors, and modulate cognate interaction (and thereby thyrotropic activity). TSHR sequences available from two life orders, teleost and mammals, were analyzed. Teleost TSHRs with low affinity are expressed in many nonthyroidal tissues and show a tendency to gene duplication. In some teleosts, TSHR has limited specificity, and in others extremely high constitutive activity, suggesting the possibility of ligand-independent receptor function. Although mammalian TSHR, in contrast to other glycoprotein hormone receptors, maintains relatively high constitutive activity, the thyrotropic activity of TSH appears to decline in hominoids including humans, probably as part of metabolic adaptation to the changing environment. Critical TSHR residues that determine hormone specificity have been identified in the leucine-rich repeats, and others within the cysteine-rich C-flanking region that determines hormonal activation as well as receptor silencing. Transmembrane (TM) helices, particularly the TM5 and TM6, are likely involved in receptor homodimerization and a unique motif in TM7 appears essential to receptor silencing and internalization. Surprisingly, ternary structures in the intracellular domain as opposed to specific sequence motifs are critical for intracellular TSHR trafficking. It is evident that progress in understanding structure-function relationships of TSHR and its ligand can be further stimulated by inclusion of evolutionary analysis of their primary, secondary and tertiary structure. Such an integrated approach should also contribute to the rational design of highly efficacious therapeutics with either agonistic or antagonistic properties.

The genetics of autoimmune thyroid disease.

Autoimmune thyroid diseases (AITDs), such as Graves' hyperthyroidism, are common disorders involving multiple genes and the environment. Some pathogenetic genes are probably shared between these diseases and non-endocrine autoimmune diseases, whereas others are disease specific. Population studies show that major histocompatibility complex alleles and CTLA4 confer risk for AITDs. Genetic studies have identified over 20 potential loci; only one, mapping to 5q31, has been convincingly replicated. Despite its recent emergence as an autoimmunity gatekeeper gene, linkage of CLTA4 to AITDs was described in only one Caucasian population subset. Like in the case of many complex genetic disorders, identifying AITD pathogenetic genes is limited by the ability of data analysis methods to discern the influence of genes of minor effect in a relatively small database.

Evolutionary divergence of thyrotropin receptor structure.

The availability of 18 thyrotropin receptor (TSHR) sequences, including two recent entries for primates and seven from fish, have allowed us to investigate diversification of residues or domains during evolution. We used a likelihood ratio test for evolutionary rate shifts [Proc. Natl. Acad. Sci. 98 (2001) 14512] using LH/CGR sequences as an out-group. At each residue in the alignment, a statistical test was performed for a rate shift at the divergence between mammals and fish. Eighty-two rate shift sites were found, significantly more than was expected (p < 0.0001). The occurrence of rate shifts was highest in the intracellular tail, lowest in the transmembrane serpentine and intermediate in the ectodomain. In 52 mammalian sites, the rates were significantly faster than for the corresponding sites in fish. We have identified rate shift in sites important to TSHR function or in intimate proximity to such regions. The former category includes residues 53 and 55 (of LLR1 beta strand) and 253 and 255 (of LLR9 beta strand), crucial to TSH thyrotropic activity, residue 113, the site of N-linked glycosylation limited to humans, residue 310, an important switch in the hinge region for receptor binding and constitutive activity and residue 382 which centres a motif important for TSH-mediated receptor activation. The rate shifts positions close to functional region include a site proximal to a TSHR-specific motif on LLR3 beta strand, sites important in TM helix structure and homodimerization as well as, in the case of the third intracellular loop, to TSHR/G protein coupling. Rate shift analyses have identified residues whose manipulation in the human TSHR may lead to better understanding of receptor functions and help in the creation of designer analogues.

Thyroglobulin increment after thyroid hormone withdrawal is a reliable indicator for the detection of significant remnants or metastases in patients with differentiated thyroid carcinoma.

Serum thyroglobulin (Tg) measurement has a pivotal role in the management of differentiated thyroid carcinoma (DTC). Serum Tg increment after thyroid hormone discontinuation seems to be a better predictor of tumor recurrence, however, minimal Tg increment may not be a specific marker. This study tries to evaluate the importance of different levels of Tg increment after thyroid hormone discontinuation. Fifty-five patients (46 females and 9 males with mean age of 41.40 yrs) with DTC, treated with total or subtotal thyroidectomy and radioiodine-131 ((131)I) were studied. Ninety-one per cent of the patients had papillary carcinoma. Serum Tg and thyroid stimulating hormone (TSH) were measured using high sensitive IRMA assays during thyroxine (T4) suppression and after discontinuation of T4 treatment. The mean time interval between Tg on T4 and off T4 was 110.29+/-53.43 days and less than 180 days in all patients. Serum Tg level was increased >or= 1 ng/ml in 25 patients after discontinuation of T4. Of these patients, 17 had metastatic disease or a detectable thyroid remnant. Of 16 patients with unchanged Tg (-1or= 7 ng/ml had residual disease or metastases. If DeltaTg was unchanged or decreased, the negative predictive value was 83.3%. The sensitivity of WB(131)IS was 63.6% for the detection of thyroid remnant or metastases. Our study indicates that DeltaTg is a more reliable indicator of remnant disease than on T4-Tg or off T4-Tg levels.

Gene therapy of anaplastic thyroid carcinoma with a single-chain interleukin-12 fusion protein.

Anaplastic thyroid carcinoma is the most aggressive type of thyroid malignancy with a mean survival time of less than 8 months. No effective therapeutic approach is currently available, making the development of novel treatments necessary. Interleukin (IL)-12 is a proinflammatory heterodimeric cytokine with strong antitumor activity. In the present study, we investigated the potential of IL-12 gene therapy for anaplastic thyroid carcinoma in BALB/c (nu/nu) nude mice. A single-chain IL-12 fusion protein construct was created to assure equal expression of its p35 and p40 subunits. Human anaplastic thyroid carcinoma cell line ARO was stably transfected with an IL-12 expression plasmid under the control of cytomegalovirus (CMV) promoter (scIL-12/CMVpDNA). High levels of functional IL-12 (26.78 +/- 4.11 ng/ml per 10(6) cells per 48 hr) were produced by scIL-12-transfected ARO cells (ARO/IL-12). Tumorigenicity in nude mice was completely lost in scIL-12-transfected ARO cells, as demonstrated by the lack of tumor formation after subcutaneous injection of 2 x 10(6) ARO/IL-12 cells, even though there was no difference in cell proliferation between ARO and ARO/IL-12 cells. Tumor growth was observed after challenge with ARO tumor cells, indicating that protective immunity had not developed against the parental cells. Furthermore, the growth rate of established subcutaneous ARO tumors was significantly reduced by either subcutaneous injection of 2 x 10(6) ARO/IL-12 cells weekly or intramuscular injection of 50 microg scIL-12/CMVpDNA twice weekly. The antineoplastic activity of ARO/IL-12 cells was, however, abrogated by intraperitoneal injection of anti-natural killer (NK) cell antibody. Moreover, significantly higher number of ARO/IL-12 cells and ARO cells were killed by splenocytes from nude mice previously treated with ARO/IL-12 compared to those treated with ARO cells (32% vs. 9% when ARO were used as target cells, 43% vs. 17% when ARO/IL12 were used as target cells; p < 0.01) in an in vitro cytotoxicity assay. Again, tumor cell killing was neutralized by the addition of anti-NK cell antibody in the assay. In conclusion, we have demonstrated successful gene therapy with a scIL-12 fusion protein against anaplastic thyroid carcinoma in an in vivo model. The immune response against ARO/IL-12 cells is mediated by NK cells. These results may set the stage for clinical application of IL-12 gene therapy for poorly differentiated thyroid carcinoma.

Evolution of the thyrotropin receptor: a G protein coupled receptor with an intrinsic capacity to dimerize.

The rapidly escalating number of genome sequences has emphasized the basic tenants of the schema of life. By the same token comparisons according to specialized function or niche within nature expose genomic strategies to optimize the use of resources and ensure biological success. Increasing complexity may result from diversification, shuffling, and re-arrangement of an otherwise limited functional genomic complement. To further test the concept of relative structural plasticity of the TSH receptor we sequenced the TSHR gene of two Old World monkey species Macaca mulatta and Cercopithecus aethiops, evolutionary removed from Homo sapiens by >20Myr. Both genes encoded a protein of 764 residues. This structure was 99% homologous between the two species of Old World monkeys while C. aethiops was 97% and M. mulatta was 96% homologous to H. sapiens. TSHR sequence comparisons were sought for an additional eight mammals as well as four (two Salmon, Tilapia, and Sea Bass) from teleosts. The amino-acid sequences of the 14 TSH receptors were similar. The most variable sequences were those of the intracellular tail and the distal cysteine-rich C-terminus flanking region of the ectodomain, whereas the trans-membrane domain was most preserved. Some sequences were decidedly H. sapiens specific, while others were primate specific or showed the changes expected of evolutionary descent. Others, however, exhibited "cross-species polymorphism," sometimes at quite remarkable evolutionary distances. As opposed to H. sapiens the sequence differences may have subtle influences on TSHR function or may affect long-range compensation for radical changes in adducts. The two Old World monkeys share with other lower mammals the absence of a glycosylation site at 113-115. Sea Bass and Tilapia have four glycosylation sites, whereas the two salmon receptors have only three. Changes in some critical residues raise questions about variation in function: thus S281 is conserved in all mammals and an important determinant of negative agonist function of TSHR is replaced by R in Sea Bass. Likewise the K183, found at an important transitional region at LRR 6 conserved in all mammals, is represented by M in fish and may contribute to TSHR lutenization in fish. There is no evidence that evolutionary changes in primate receptors are more rapid than that in other mammals and the separation times of different mammals based on silent nucleotide changes of TSHR are closely parallel to archaeological estimates. Results of correlated mutation analysis, referenced to the rhodopsin crystal structure, affirms dimerization of TSHR transmembrane helices. In addition, it suggests the involvement of critical lipid-facing residues in the helices in receptor dimerization and oligomerization. We highlight the value of evolutionary informatics and set the stage for dissecting out potential subtle differences in TSHR function associated with structural variations.