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Written by Hai-Yang Zhang, M.D.,#1,*  Feng-Yao Wu , M.D.,#1,*  Xue-Song Li , M.D.,2  Ping-Hui Tu , M.D.,1  Cao-Xu Zhang , M.D.,1  Rui-Meng Yang , M.D., Ph.D.,1  Ren-Jie Cui , Ph.D.,1  Chen-Yang Wu , M.D.,1  Ya Fang , M.D., Ph.D.,1  Liu Yang , M.S.,1  Huai-Dong Song , M.D., Ph.D.,1 and Shuang-Xia Zhao , M.D., Ph.D.1 Ann Lab Med. 2024 Jul 1; 44(4): 343–353. Published online 2024 Mar 4.   doi:  10.3343/alm.2023.0337 Abstract Background Genetic defects in the human thyroid-stimulating hormone (TSH) receptor ( TSHR ) gene can cause congenital hypothyroidism (CH). However, the biological functions and comprehensive genotype–phenotype relationships for most TSHR  variants associated with CH remain unexplored. We aimed to identify TSHR  variants in Chinese patients with CH, analyze the functions of the variants, and explore the relationships between TSHR  genotypes and clinical phenotypes. Methods In total, 367 patients with CH were recruited for TSHR  variant screening using whole-exome sequencing. The effects of the variants were evaluated by in-silico programs such as SIFT and polyphen2. Furthermore, these variants were transfected into 293T cells to detect their Gs/cyclic AMP and Gq/11 signaling activity. Results Among the 367 patients with CH, 17 TSHR  variants, including three novel variants, were identified in 45 patients, and 18 patients carried biallelic TSHR  variants. In vitro experiments showed that 10 variants were associated with Gs/cyclic AMP and Gq/11 signaling pathway impairment to varying degrees. Patients with TSHR  biallelic variants had lower serum TSH levels and higher free triiodothyronine and thyroxine levels at diagnosis than those with DUOX2  biallelic variants. Conclusions We found a high frequency of TSHR  variants in Chinese patients with CH (12.3%), and 4.9% of cases were caused by TSHR biallelic variants. Ten variants were identified as loss-of-function variants. The data suggest that the clinical phenotype of CH patients caused by TSHR  biallelic variants is relatively mild. Our study expands the TSHR  variant spectrum and provides further evidence for the elucidation of the genetic etiology of CH. Keywords: Congenital hypothyroidism, Recessive inheritance, Thyroid-stimulating hormone receptor, Variant, Whole-exome sequencing Introduction Congenital hypothyroidism (CH) is a disease characterized by impairments in neurodevelopment and physical growth and development owing to dysfunction of the hypothalamic-pituitary-thyroid axis present at birth [ 1 ]. CH is the most common congenital endocrine metabolic disease, with a global prevalence of 1/2,000–1/3,000 [ 1 ]. With the recent developments in gene sequencing technologies, an increasing number of pathogenic genes related to CH, including genes related to thyroid dysgenesis and dyshormonogenesis, have been reported. Among these, the thyroid-stimulating hormone (TSH) receptor ( TSHR ) gene is one of the widely investigated candidate genes [ 2 , 3 ]. The human TSHR gene is located on chromosome 14q31 and encodes a G-protein-coupled receptor that consists of a seven-transmembrane domain (TMD) and a large extracellular domain (ECD) responsible for high-affinity hormone binding. The TSHR is activated upon binding to TSH and induces two signal transduction pathways: the Gs/cyclic AMP (cAMP) pathway and the Gq/11 phospholipase C pathway, which contribute to thyroglobulin iodination and cell proliferation, whereas the Gs pathway is also responsible for iodine uptake regulation in thyrocytes [ 4 , 5 ]. Both pathways are important for thyroid hormone synthesis and thyroid development [ 4 , 6 ]. Loss-of-function (LOF) variants in the TSHR can cause TSH resistance, which leads to congenital nongoitrous hypothyroidism 1 (OMIM: 275200), which presents a broad spectrum of phenotypes, ranging from severe congenital hypothyroidism to mild euthyroid hyperthyrotropinemia [ 2 , 7 - 10 ]. These LOF variants may result in a thyroid gland of normal position and size or in thyroid dysgenesis [ 11 ]. LOF variants in TSHR  were first described in patients with TSH resistance in 1995 [ 12 ]. Up to April 2021, 202 TSHR variants have been reported and documented in the Human Gene Mutation Database (HGMD). However, the biological functions of most TSHR  variants remain unknown, and genotype–phenotype relationships have not yet been clearly established. We previously identified 15 TSHR  variants in 13 out of 220 Chinese patients with CH [ 13 ]. In the present study, we enrolled an additional 367 patients with CH, expanding the sample for screening TSHR  variants and characterizing the phenotypes of patients with CH carrying TSHR  variants. The biological functions of the variants were investigated through a series of in vitro  experiments. We expected this study to deepen our understanding of the genetic landscape and functional consequences of TSHR  variants and to provide valuable insights into the clinical management of patients harboring TSHR  variants. Materials and methods Patients In total, 367 patients were enrolled from the Chinese Han populations in Jiangsu province, Fujian province, Anhui province, and Shanghai. Among them, 362 patients (98.7%) received neonatal CH screening, which was performed using filter-paper blood spots (obtained through a heel prick) within 3–5 days after birth. Patients with TSH levels ≥10 μIU/mL at initial screening were recalled for re-examination using an immune-chemiluminescence assay (UniCelDxI 800; Beckman, Indianapolis, IN, USA) to determine the levels of TSH, free triiodothyronine (FT3), and free thyroxine (FT4). The details of the diagnostic standards for CH have been described in our previous study [ 14 ]. In addition, five patients who were on l-thyroxine replacement therapy were recruited from outpatient clinics. Although these patients were not neonatally screened, they had a clear history of CH. Thyroid morphology was determined by experienced radiologists through thyroid ultrasound or technetium-99m scanning. Written informed consent to participate was provided by the participants’ legal guardians, and the study was approved by the Ethics Committee of Shanghai Ninth People’s Hospital affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai, China (approval number: 2016-76-T33). Whole-exome sequencing (WES) WES was performed as previously described [ 15 ]. Genomic DNA was extracted from peripheral blood, fragmented to 200–300 bp, and ligated to adapters using the KAPA HyperPrep Kit (Roche, Basel, Switzerland). Exonic hybrid capture was performed according to the instructions in the Roche SeqCap EZ Library SR User’s Guide. Library quality and levels were determined using the MAN CLS140145 DNA 1 K Chip (PerkinElmer, Waltham, MA, USA) and the PE LabChip GXII Touch (PerkinElmer, Waltham, MA, USA). The Illumina HiSeq 3000 system (Illumina, San Diego, CA, USA) was then used to sequence the paired-end libraries with 150-bp paired-end reads, averaging approximately 100× depth. Statistical analysis IBM SPSS Statistics version 25.0 (IBM Corp., Armonk, NY, USA) was used for all statistical analyses. Quantitative variables are presented as mean±SE. The normality of the data was assessed using the Shapiro–Wilk test and intergroup comparisons were performed using Student’s unpaired t -test (for normally distributed data) or the Mann–Whitney U test (for non-normally distributed data), as appropriate. Categorical variables are presented as percentages and were compared using the chi-square test or Fisher exact test, as appropriate. P <0.05 was considered statistically significant. Additional methods are available in the Supplemental Materials . Results Clinical characteristics of 367 patients with CH In total, 367 patients with CH, including 196 boys and 171 girls, were recruited in this study. The mean serum FT3, FT4, and TSH levels at diagnosis were 4.87 pmol/L, 10.55 pmol/L, and 77.57 μIU/mL, respectively (reference ranges: FT3, 3.85–6.01 pmol/L; FT4, 7.46–21.11 pmol/L; TSH, 0.34–5.60 μIU/mL). Based on the serum FT4 level at diagnosis, CH was classified as severe (FT4<5 pmol/L), moderate (5 pmol/L≤FT4<10 pmol/L), or mild (FT4≥10 pmol/L) [ 16 ]. In the present cohort, 49.1% of patients had moderate or severe CH, and 50.9% of patients presented with mild CH. There were no significant differences in hormone levels and other clinical characteristics between boys and girls ( Table 1 ). Screening for TSHR  variants in Chinese patients with CH and pedigree analysis Among the 367 patients with CH, 45 patients carried 17 non-synonymous TSHR  variants, including 16 missense variants and one nonsense variant. The TSHR variant frequency was 12.3% (45/367). Out of 17 variants, three (p.S237G, p.W546C, and p.M728T) were first reported in this study, and 10 were recurrent variants (p.G132R, p.G245S, p.S305R, p.N432S, p.R450H, p.F525S, p.R609X, p.Y613C, p.V689G, and p.E758K). p.R450H, which is a hotspot variant in the Chinese population, had the highest frequency (2.7%) ( Table 2 ). Four of the 17 variants were located in the leucine-rich repeat (LRR) domain of the TSHR protein ( Fig. 1A ). Conservation analysis of the three novel variants showed that p.W546C was highly conserved across species, whereas p.M728T was less conserved ( Fig. 1B ). Out of the 45 patients with TSHR  variants, 18 patients carried biallelic variants. We conducted a long-term follow-up of two patients with TSHR biallelic variants (CHT558 and CHT573) and collected blood samples from their parents for pedigree analysis. Sanger sequencing showed that the biallelic variants carried by the patients were inherited from their father and mother separately, which is in line with an autosomal recessive inheritance pattern ( Fig. 2 ). Pathogenicity prediction of detected TSHR  variants The potential effects of the 17 variants identified were assessed using in silico programs (SIFT, Polyphen-2, Mutation Taster, and M-CAP). All four programs predicted that the variants p.I216T, p.G245S, p.A275T, p.N432S, p.R450H, p.A526T, p.R531W, p.W546C, p.Y613C, and p.V689G were detrimental to TSHR protein function and that the novel p.M728T variant was harmless. The prediction results of the other variants were inconsistent among the four programs ( Supplemental Data Table S1 ). Subsequently, we predicted the three-dimensional structure of the wild-type (WT) and three novel mutant proteins using in silico tools. In the novel variant p.S237G, a polar neutral serine was replaced with a non-polar aliphatic glycine, disrupting the hydrogen bond between serine 237 and lysine 211 ( Supplemental Data Fig. S1A ). For the p.W546C variant, the aromatic tryptophan residue at 546 was mutated to a neutral cysteine, disrupting the hydrogen bond between tryptophan 546 and asparagine 455, destabilizing the helix ( Supplemental Data Fig. S1B ). As for p.M728T, the model confidence at amino acid 738 was very low; therefore, it was not analyzed. The American College of Medical Genetics (ACMG) issued new guidelines for the interpretation of sequence variants in 2015, describing a process for classifying variants into five categories based on criteria related to typical variant evidence types (such as population data, computational data, functional data, and segregation data). Variants are classified as pathogenic (P), likely pathogenic (LP), variants of uncertain significance (VUS), likely benign (LB), or benign (B) [ 17 ]. Based on the available evidence, the pathogenicity of the 17 variants identified was classified according to the ACMG guidelines and standards. Five variants (p.G132R, p.N432S, p.R450H, p.F525S, and p.R609X) were classified as P or LP, and p.M728T was classified as LB. The remaining 11 variants were classified as VUS ( Supplemental Data Table S1 ). Clinical characteristics of CH patients with TSHR  variants The clinical phenotypes of the 45 CH patients with TSHR  variants were compared with those of the 322 CH patients without TSHR variants. There were no significant differences between the two groups in terms of hormone levels, age at diagnosis, and initial levothyroxine dose ( Supplemental Data Table S2 ). Thyroid functional information at diagnosis was collected for 25 out of 45 patients who harbored TSHR  variants, including four patients with severe CH, seven with moderate CH, and 14 with mild CH. One patient (CHT241) harboring TSHR  variants had thyroid dysgenesis ( Supplemental Data Table S3 ). Among the seven patients with TSHR biallelic variants, only one patient showed moderate CH, and the remaining six patients presented with mild CH. However, patients with the TSHR  monoallelic variant can present with mild to severe CH. Surprisingly, we found that the patients with the TSHR  monoallelic variant had more severe hypothyroidism, with lower FT4 levels (9.58±1.50 vs. 15.87±1.18, P =0.020) at diagnosis, than patients with TSHR  biallelic variants ( Fig. 3A–3C ). Dual oxidase 2 ( DUOX2 ) is a key protein for thyroid hormone synthesis, and DUOX2  is the most frequently mutated gene in Chinese patients with CH [ 14 , 18 ]. We compared the clinical characteristics of patients with TSHR  or DUOX2  biallelic variants in the present cohort. Compared with CH patients with DUOX2  biallelic variants, patients with TSHR  biallelic variants had lower serum TSH levels and higher FT3 and FT4 levels at diagnosis (TSH: 52.96±17.84 vs. 105.77±5.48, P =0.012; FT3: 5.71±0.43 vs. 4.47±0.15, P =0.025; FT4: 15.87±1.18 vs. 7.20±0.45, P <0.001) ( Fig. 3D–3F ). In patients with DUOX2  variants, hypothyroidism may vary with age, whereas in patients with TSHR variants, it tends to remain stable over time. Therefore, we compared thyroid function at 6 months and 3 yrs of age between patients carrying TSHR  biallelic variants and those carrying DUOX2  biallelic variants. Interestingly, patients harboring TSHR  biallelic variants exhibited higher FT4 levels at both 6 months and 3 yrs of age than patients carrying DUOX2  biallelic variants (6 months: 20.60±1.27 vs. 16.77±0.65, P =0.041; 3 yrs: 22.72±1.73 vs. 16.83±0.96, P =0.019) ( Supplemental Data Fig. S2 ). Functional assessment of the TSHR  variants in vitro The eight variants (p.G132R, p.I216T, p.G245S, p.N432S, p.R450H, p.F525S, p.A526T, and p.V689G) detected in 18 patients with TSHR biallelic variants and the three novel variants (p.S237G, p.W546C, and p.M728T) were selected for molecular function assessment. The variants were transiently transfected into 293T cells, and Gs/cAMP and Gq/11 signal transduction were investigated by measuring cAMP levels and luciferase activity, respectively. Compared with 293T cells transfected with the WT plasmid, cAMP production in response to bovine TSH (bTSH) was significantly reduced in cells transfected with the p.G132R, p.I216T, p.S237G, p.G245S, p.N432S, p.R450H, p.F525S, p.A526T, and p.W546C mutant plasmids. However, the p.V689G and p.M728T variants did not affect cAMP production ( Fig. 4A ). The p.G132R, p.I216T, p.S237G, p.G245S, p.F525S, p.A526T, p.W546C, and p.V689G variants showed partial Gq/11 signaling activity (14%–57%), whereas activity was almost abrogated for the p.N432S and p.R450H variants (<10%) after stimulation with 100 U/L bTSH. The p.M728T variant had no effect on Gq/11 signaling ( Fig. 4B ). We next investigated the protein expression and subcellular localization of the three novel variants (p.S237G, p.W546C, and p.M728T) in 293T cells. Western blot analysis showed no significant differences in protein expression between the WT and the three mutants ( Supplemental Data Fig. S3A and 3B ). Subcellular localization analysis showed that the WT and three mutant TSHR proteins all localized to the cell membrane in an intact manner ( Supplemental Data Fig. S3C ). Discussion Through comprehensive screening, we identified 17 distinct TSHR  variants in 367 CH patients in China. We found a high frequency of TSHR  variants in Chinese patients with CH (45/367, 12.3%), with 4.9% of patients carrying biallelic TSHR variants. We identified three novel variants (p.S237G, p.W546C, and p.M728T), two of which (p.S237G and p.W546C) impaired TSHR biological functions in the Gs/cAMP and Gq/11 pathways. Seventeen non-synonymous TSHR variants were identified in 45 CH patients, with a detection rate of 12.3%, which is higher than the rates reported in most domestic studies [ 13 , 19 - 21 ] but lower than those in two cohort studies in Italy and Korea [ 22 , 23 ]. Most TSHR  LOF variants reported to date are located in exons 1, 4, 6, and 10 [ 11 ]. In this study, 12 of the 17 identified variants were located in exon 10, whereas none were located in exons 1, 4, and 6. These findings suggest that there may be regional and ethnic differences in the spectrum of TSHR  variants. In addition, we found a hotspot variant, p.R450H, which is one of the most common TSHR  LOF variants and has been demonstrated to have a founder effect in Japan [ 24 ]. Notably, among the 45 patients carrying TSHR  variants, 18 carried biallelic variants. The total residual Gs/cAMP and Gq/11 pathway signaling activities in CH patients with TSHR  biallelic variants were calculated as the sum of pathway signaling activities from both TSHR variant alleles divided by two. Two patients (CHT385 and CHT573) harboring the p.R450H homozygous variant, who had 35% Gs/cAMP signaling pathway activity and 6% Gq/11 signaling pathway activity, had clinically similar phenotypes and presented with mild hypothyroidism. Patient CHT506, who carried the p.G132R homozygous variant, had a 62% reduction in Gs/cAMP signaling pathway activity and 70% Gq/11 signaling pathway activity and was diagnosed as having moderate CH, with serum TSH and T4 levels of 150.00 μIU/mL and 9.27 pmol/L, respectively. Patient CHT436, who harbored the p.N432S/p.R450H biallelic variants with residual Gs/cAMP and Gq/11 signaling pathway activities of 32% and 8%, respectively, presented with mild CH, with serum TSH and FT4 levels of 27.26 μIU/mL and 18.66 pmol/L. Patients CHT445 and CHT516 carried the p.G132R/p.R450H biallelic variants, with residual Gs/cAMP and Gq/11 signaling pathway activities of 37% and 18%, respectively. They both exhibited mild CH. Patient CHT553 harboring the p.R450H/p.F525S biallelic variants had 30% and 32% residual Gs/cAMP and Gq/11 signaling pathway activities, respectively, and was diagnosed as having mild CH, with serum TSH and FT4 levels of 25.56 μIU/mL and 15.96 pmol/L, respectively ( Supplemental Data Table S4 ). These functional experimental results in patients with TSHR  biallelic variants support the hypothesis that TSHR  variants can cause the onset of CH. Pedigree analysis of two patients showed that CH caused by TSHR  variants is inherited in an autosomal recessive manner, which is consistent with previous findings [ 12 , 25 , 26 ]. LOF TSHR variants result in variable TSH resistance manifested as euthyroid hyperthyrotropinemia with a normal thyroid gland (fully compensated TSH resistance), mild hypothyroidism with a normal thyroid gland (partially compensated TSH resistance), or severe hypothyroidism with thyroid dysgenesis (uncompensated TSH resistance) [ 2 ]. In the present study, out of seven patients with TSHR biallelic variants, six patients presented with mild hypothyroidism, and only one patient presented with moderate hypothyroidism. Moreover, patients with TSHR  biallelic variants had milder hypothyroidism than those with DUOX2  biallelic variants. These findings indicate that the phenotypes of CH caused by TSHR  defects are milder and associated with completely or partially compensated TSH resistance. The TSHR is a G-protein-coupled receptor with a TMD domain and a large ECD, which comprises an LRR domain involved in hormone binding specificity and a hinge region, linking the LRR domain to the TMD [ 11 , 27 ]. TSHR activation results in intracellular signaling via the Gs protein, which leads to cAMP cascade activation, and via the Gq protein, which leads to phospholipase C cascade activation. In the present study, four variants (p.G132R, p.I216T, p.S237G, and p.G245S) were located in the LRR domain, and they caused varying degrees of impairment to the Gs/cAMP and Gq/11 signaling pathways. The novel p.S237G variant had no effect on the expression and membrane localization of the TSHR protein but partially hindered Gs/cAMP and Gq/11 signaling. This may be attributed to the replacement of amino acids altering the TSHR protein structure, thereby decreasing its ability to bind to TSH. The novel p.W546C variant, located in the fourth TMD of TSHR, is highly conserved among species. In silico  tools predicted that this missense variant is detrimental to protein stability and function. In vitro experiments demonstrated that the p.W546C variant damages receptor function by affecting the Gs/cAMP and Gq/11 signaling pathways. p.M728T, another novel variant identified in the present study, is located in the C-terminal intracellular region of TSHR. Chazenbalk, et al . [ 28 ] confirmed that the removal of the C-terminal 2/3 residues (Q709–L764) of TSHR did not impair receptor function. Concurrently, functional experiments in the present study showed that the p.M728T variant did not interfere with the Gs/cAMP or Gq/11 pathway. Numerous studies have confirmed that the hotspot variant p.R450H not only results in reduced cAMP activity and severely impaired Gq/11 pathway activity but also reduces the TSH binding ability of TSHR [ 5 , 13 , 24 , 29 ], which is consistent with our findings. The phenotypes of the TSHR  monoallelic variant are reportedly always mild, whereas biallelic variants are often associated with a more severe phenotype [ 7 ]. However, we found that patients with the TSHR monoallelic variant presented with mild to severe CH. Therefore, we compared thyroid function at diagnosis in patients with monoallelic and biallelic TSHR  variants. Surprisingly, patients with the TSHR monoallelic variant had lower FT4 levels, which may be because of the following reasons. First, in our cohort, 12 of 27 patients with the TSHR  monoallelic variant harbored biallelic variants in 21 other CH pathogenic genes ( NKX2-1 , NKX2-5 , FOXE1 , PAX8 , HHEX , TPO , SLC5A5 , TG , DUOX2 , DUOXA2 , TSHR , SLC26A4 , IYD , DIO1 , DIO2 , THRA , THRB , DUOX1 , DUOXA1 , GNAS , and SLC16A2 ) as described in our previous study [ 14 ] ( Supplemental Data Table S3 ). Compared with patients with TSHR  biallelic variants, patients with oligogenic variants, including in TSHR , had lower FT4 levels and higher TSH levels at diagnosis, whereas patients with only a TSHR  monoallelic variant showed no difference in thyroid function at diagnosis ( Supplemental Data Fig. S4 ), which partially explains why patients with the TSHR  monoallelic variant presented more severe hypothyroidism than those with TSHR  biallelic variants. Second, the genetic etiology of CH is largely unknown, and patients with the TSHR monoallelic variant may also carry novel CH-causative genes, leading to a more severe phenotype. Finally, environmental modifiers, such as iodine intake and ethnicity, should be considered in addition to genetic factors to explain this phenotypic variation. For example, Vigone, et al . [ 30 ] reported phenotypic differences between two brothers harboring the same genetic variants attributed to different neonatal iodine supplies, which suggested that the different neonatal iodine supplies acted as disease modifiers. This study had some limitations. First, among the 18 patients carrying biallelic TSHR  variants, pedigree analysis was conducted for only two families. Second, we did not clarify whether patients carrying TSHR variants require lifelong thyroxine therapy. Third, the pathogenic mechanism related to the presence of a heterozygous sequence variant in TSHR in patients with CH was not fully identified. In future work, we will mine and analyze unknown pathogenic genes in CH to gain insight into the molecular mechanisms of CH pathogenesis. In conclusion, we reported 17 TSHR variants in 367 Chinese patients with CH and investigated the biological function of 11 variants (eight biallelic and three novel variants). Two novel variants (p.S237G and p.W546C) impair TSHR protein biological function by interfering with Gs/cAMP and Gq/11 signaling. Characterization of the phenotypes of patients with TSHR  variants revealed that TSHR  biallelic variants cause mild CH. The present study expanded the TSHR  variant spectrum and provided further evidence for the elucidation of the genetic etiology of CH. References 1. van Trotsenburg P, Stoupa A, Léger J, Rohrer T, Peters C, Fugazzola L, et al. 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Written by Mst. Noorjahan Begum, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing, 1 , 2 , 3   Rumana Mahtarin , Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing, 1 , 4   Md. Tarikul Islam , Formal analysis, Investigation, 1   Sinthyia Ahmed , Formal analysis, Investigation, Methodology, 5   Tasnia Kawsar Konika , Investigation, 6   Kaiissar Mannoor , Conceptualization, Supervision, Writing – review & editing, 1   Sharif Akhteruzzaman , Conceptualization, Supervision, Writing – review & editing, 2  and Firdausi Qadri , Conceptualization, Supervision 1 , 7 ,* Dhanusha Yesudhas, Editor PLoS One. 2023; 18(8): e0282553. Published online 2023 Aug 10.   doi:  10.1371/journal.pone.0282553 Abstract The disorder of thyroid gland development or thyroid dysgenesis accounts for 80–85% of congenital hypothyroidism (CH) cases. Mutations in the TSHR gene are mostly associated with thyroid dysgenesis, and prevent or disrupt normal development of the gland. There is limited data available on the genetic spectrum of congenital hypothyroid children in Bangladesh. Thus, an understanding of the molecular aetiology of thyroid dysgenesis is a prerequisite. The aim of the study was to investigate the effect of mutations in the TSHR  gene on the small molecule thyrogenic drug-binding site of the protein. We identified two nonsynonymous mutations (p.Ser508Leu, p.Glu727Asp) in the exon 10 of the TSHR gene in 21 patients with dysgenesis by sequencing-based analysis. Later, the TSHR368-764 protein was modeled by the I-TASSER server for wild-type and mutant structures. The model proteins were targeted by thyrogenic drugs, MS437 and MS438 to perceive the effect of mutations. The damaging effect in drug-protein complexes of mutants was explored by molecular docking and molecular dynamics simulations. The binding affinity of wild-type protein was much higher than the mutant cases for both of the drug ligands (MS437 and MS438). Molecular dynamics simulates the dynamic behavior of wild-type and mutant complexes. MS437-TSHR368-764MT2 and MS438-TSHR368-764MT1 showed stable conformations in biological environments. Finally, Principle Component Analysis revealed structural and energy profile discrepancies. TSHR368-764MT1 exhibited much more variations than TSHR368-764WT and TSHR368-764MT2, emphasizing a more damaging pattern in TSHR368-764MT1. This genetic study might be helpful to explore the mutational impact on drug binding sites of TSHR protein which is important for future drug design and selection for the treatment of congenital hypothyroid children with dysgenesis. 1. Introduction Congenital hypothyroidism is associated with various factors including genetics. Genetic causes account for about 15 to 20 percent of congenital hypothyroidism (CH). Although CH is a genetically heterogeneous disorder, the candidate genes divide the disorder into two main groups namely thyroid dysgenesis and thyroid dyshormonogenesis. Different studies including online databases such as Genetics Home Reference and Online Mendelian Inheritance in Men (OMIM) suggested that about 10–20 percent of total cases with CH were associated with thyroid dyshormonogenesis that would result from mutations in one of several genes involved in the biosynthesis of thyroid hormones [ 1 ]. The above-mentioned databases also described that about 80–85% of CH cases are associated with disorders of thyroid gland development (Dysgenesis) which is categorized as ectopic (located in a distant region, 40%), agenesis (absent of thyroid gland, 40%), and the other cases are accompanying with hypoplasia (small size). Although the actual cause of thyroid dysgenesis is still under investigation, some studies have suggested that 4 major genes that play roles in the proper growth and development of the thyroid gland, such as TSHR  (Thyroid 3 stimulating hormone receptor) and three transcription factors- TTF-1 , TTF-2 , and PAX8  (paired box-8, transcription factor) [ 2 ]. Mutations in these genes prevent or disrupt the normal development of the gland. The TSHR  gene is predominantly related to thyroid dysgenesis, as most of the mutations occurs in the gene in CH patients [ 2 ]. TSHR is a G protein-coupled transmembrane receptor which is present on the surface of thyroid follicular cells. TSH, secreted by the anterior pituitary, mediates its effect through TSHR which is crucial for thyroid gland development and function. The TSHR  gene is located on chromosome 14q31 and contains 11 exons code for a receptor protein of 764 amino acid residues [ 3 , 4 ]. TSHR has high affinity binding sites for TSH. Mutations in the TSHR gene result in mutant TSHR protein which lacks its binding affinity to TSH or loses its ability to activate adenylate cyclase. Thus, mutant TSHR protein disrupts thyroid gland development and proper functioning. TSHR  mutation may also be present in a normally placed thyroid gland. TSHR gene mutation is reported to be inherited as an autosomal recessive manner and exon 10 is known to carry the majority of the mutations [ 5 ]. In a high-throughput screening system, two small molecule agonists (MS437 and MS438) exhibited pharmacotherapeutic potential with the highest potency (EC50 of 13x10-8 M, and EC50 of 5.3x10-8 M respectively) [ 6 ]. Very limited data are available on genetic study of Bangladeshi hypothyroid patients. Therefore, the present study tried to explore the effect of two non-synonymous mutations in the 3D structure of TSHR368-764 targeted by thyrogenic drugs, MS437 and MS438 which will help to update any future treatment strategy including suitable drug design for Congenital Hypothyroid children. 2. Methods and materials 2.1. Study design, clinical settings, and ethical clearance The study was designed and carried out on 21 confirmed cases of Congenital Hypothyroid children with dysgenesis who were kept under treatment of Levothyroxine (LT4) drug in the Department of Endocrinology and National Institute of Nuclear Medicine and Allied Sciences (NINMAS) of Bangabandhu Shaikh Mujib Medical University (BSMMU). Ethical permission was obtained from the Ethical Review Committee of University of Dhaka (CP-4029) and the study was collaborated with NINMAS and Dept. of Endocrinology, BSMMU for specimen collection. Prior to enrollment of study participants, a written informed consent along with the clinical information was collected from the parent(s) or legal guardian(s) of each patient. 2.2. Collection and processing of blood specimens Blood Specimens were collected from the participants to conduct the molecular, biochemical and metabolic profiling tests. A total of 3ml blood was collected from each participant. All the samples were transported to the laboratory immediately. After the genomic DNA isolation, EDTA containing blood was stored at -70°C freezer. 2.3. Molecular analysis of TSHR  gene Now-a-days, gene-based study plays the key role to explore the actual cause of a particular disease. The present study was designed to perform the molecular analysis in various steps. 2.3.1. Genomic DNA isolation to perform PCR Genomic DNA was isolated from the EDTA blood by using Qiagen DNAeasy mini kit according to the manufacturer’s instruction. 500 μl of FG1 buffer was taken in a 1.5 ml microcentrifuge tube. 200 μl of whole blood was added to the FG1 buffer and mixed by inverting the tube 5–10 times. The mixture was then centrifuged at 10,000×g for 5 minutes in fixed angle rotor. The supernatant was carefully removed so that the pellet remained in the tube. 1μl of QIAGEN protease was added to 100 μl of FG2 buffer and mixed by vortex in a fresh Eppendorf tube. Then 100 μL of FG2/QIAGEN protease was added to the pellet and vortexed immediately until the pellet was completely dissolved and the color was changed into olive green so that all the protein components were degraded. The mixture was then incubated in a water bath or heat block at 65°C for 5 minutes. After incubation, 100 μl of isopropanol (100%) was added and mixed by inversion until DNA was precipitated as visible threads. The tube was centrifuged for 5 minutes at 10000×g. The supernatant was discarded and the pellet was dried by keeping the tube inverted state on a clean tissue paper for one minute. 100 μl of 70% ethanol was added and vortexed for 5 seconds. The tube was centrifuged for 5 minutes at 10000×g. The supernatant was carefully aspirated using a micropipette and keeping the micro-centrifuge tube in the inverted state on the tissue paper to allow the pellet to air dry for at least 5 minutes. Over-drying was avoided as the process can make it difficult to dissolve the DNA. Depending on the pellet size, 25–50 μl of nuclease free water was added and the tube was vortexed for 5 seconds and the mixture was incubated at 65°C for one hour in water bath for dissolving DNA or left overnight at room temperature. Finally, the concentration and the purity of the DNA was measured using a Nano drop machine and adjusted the concentration for PCR. 2.3.2. Polymerase Chain Reaction (PCR) amplification of TSHR gene The isolated DNA was then amplified by PCR using TSHR gene-specific primers. At first, we performed PCR using primers set that could flank the sequence between exon 1 to exon 10, since global data showed that most of the common mutations in the TSHR  gene of the patients with Congenital Hypothyroidism were confined in this region. Next, we conducted PCR for other regions of the TSHR  gene. The primer sequences are listed in the Table 1  as follows. To amplify the desired target sequence of TSHR gene, PCR amplification was conducted on a thermal cycler (Bio-Rad, USA). The final reaction volume was 10 μl for each of the reactions which contained 1 μL 10X PCR buffer, 0.3 μL 25mM MgCl2, 2 μL 5X Q-solution, 1.6 μL 2.5 mM dNTPs mixture, 0.2 μL 10mM Forward primer and 0.2 μL Reverse primer, 0.05 μL Taq DNA Polymerase, 50 ng of genomic DNA and total reaction volume was made up to 10μL by addition of nuclease free water. The thermal cycling condition included (a) initial denaturation at 95°C for 5 minutes, (b) cyclic denaturation at 95°C for 40 seconds and annealing at 58°C for 35 seconds and extension at 72°C for 40 seconds; and (c) final extension at 72°C for 5 minutes for 35 cycles. 2.3.3. Sanger Sequencing of PCR products Before sequencing, the PCR products were purified using a Qiagen PCR purification kit (Qiagen) following manufacturer’s instruction. The cycle sequencing PCR was then performed by BigDye Chain Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems, USA) applying manufacturer’s instructions. The thermal cycling profile comprised (i) initial denaturation at 94°C for 1 minute, (ii) 25 cycles of denaturation at 94°C for 10 seconds, annealing at 58°C for 5 seconds and extension at 60°C for 4 minutes, and (iii) final extension at 60°C for 10 minutes. After cycle sequencing PCR, the products were purified using BigDye XTerminator® Purification Kit (Applied Biosystems). Then, sequencing of the purified cycle sequencing products was executed on the ABI PRISM 310 automated sequencer (Applied Biosystems, USA) [ 7 ]. 2.3.4. Sequencing data analysis The Sequencing data were obtained from ABI PRISM 310 data collection software version 3.1.0. FASTA format of sequencing data were utilized to identify mutations in the TSHR  gene by alignment with the reference sequence (Accession number; NG_009206.1 retrieved from the NCBI database) through the basic local alignment search tool (BLAST). The nucleotides sequence was converted into corresponding amino acids by ExPASy translate tool [ 7 ]. 2.4. Prediction of 3D structure of TSHR protein and ligand selection After performing the Sanger Sequencing, we detected two mutations, one was in the transmembrane (TM)-domain and another was in cytoplasmic (CT)-domain of TSHR protein. TSHR protein is composed of a total of 764 amino acids where the TM-domain and CT-domain belong to 368 to 764 amino acids of the full length TSHR protein. I-TASSER server was used to predict the 3D structures of wildtype and mutant TSHR protein (TM and CT domains) due to lack of the full-length experimental structure. I-TASSER provided the five models for TSHR368-764 based on the detected template rhodopsin x-ray crystal structure (PDB:1F88) [ 6 ] by LOMETS (local meta-threading-server) from the PDB library [ 8 ]. Target-template alignment was also provided for each model structure. On the basis of Confidence score (C-score), template modelling (TM) score, root mean square deviation (RMSD) score, and target-template aligned structures, we obtained best model for each structure. The I-TASSER predicted structure was compared with AlphaFold predicted structure by TM-align algorithm which detects the best structural alignment ( https://zhanggroup.org/TM-align/ ) [ 9 ]. Two promising small molecule ligands (MS437 and MS 438) were selected which act as agonist to TSHR protein. Since among the small molecules MS437 interacts with threonine 501 (T501), and MS438 interacts with residues serine 505 (S505) and glutamic acid 506 (E506) bind to the intrahelical region of TM3 of TSHR protein [ 6 ], we targeted the region to see the effect of mutations on that particular site. 2.5 Molecular docking, protein-ligand interactions, and molecular dynamics (MD) simulation Finally, the molecular docking was performed for I-TASSER predicted and AlphaFold predicted wild-type and mutant proteins using PyRx software [ 10 , 13 , 14 ]. Grid box center was x = 72.5922; y = 72.4245; z = 72.6927 and Grid box size was 25 in every axis during docking encompassing the active site residues Thr134 (501) for MS437, and Ser138 (505) and Glu139 (506) for MS438 in the intrahelical region of TM3 of TSHR protein. The binding affinity for both I-TASSER predicted and AlphaFold predicted structures was analyzed using PyRx software and PRODIGY [ 11 , 12 ]. Non-covalent interactions were also observed both for of MS437 and MS438 molecules using BIOVIA Discovery Studio version 4.5. The MD simulation was implemented through YASARA suits [ 13 ] employing AMBER14 force field [ 14 ] for calculations. The membrane was built during simulation. YASARA scanned the plausible transmembrane region comprising hydrophobic residues among the secondary structure elements of proteins. The protein was projected to the membrane, YASARA presented the recommended membrane insertion with required size (69.2 Å × 7.3 Å) containing phosphatidyl-ethanolamine, -choline, and -serine lipid constituents. The whole simulation system was equilibrated for 250 ps. During the equilibration phase, membrane was artificially stabilized. The entire environment was equilibrated at 310K temperature with 0.9% NaCl and water solvent. The temperature was controlled by Berendsen thermostat process during simulation. The particle Mesh Ewald algorithm maintained long-range electrostatic interactions. The periodic boundary condition was applied for the whole simulation. The time step was 1.25 fs during 50 ns MD simulation. The snapshots were collected at every 100 ps [ 12 ]. Diverse data containing root mean square deviation (RMSD), root mean square fluctuation (RMSF), total number of hydrogen bonds, radius of gyration, solvent-accessible surface area (SASA), and molecular surface area (MolSA) were retrieved from MD simulations, following our earlier MD data analysis [ 13 , 14 ]. 2.6. Principal Component Analysis (PCA) MD simulation data were applied to explore the structural and energy variabilities via principal component analysis (PCA) among TSHR-small molecule ligand (MS437 and MS438) complexes. The different multivariate energy factors were employed to explore the existing variability in the MD trajectory applying the low-dimensional space [ 13 ]. The variables from MD data were bond distances, bond angles, dihedral angles, planarity, van der Waals energies, and electrostatic energies considered for the structural and energy factors [ 14 ]. The data pre-processing were implemented by centering and scaling. In the analysis, final 45 ns MD trajectories were applied for exploration of the variations. The PCA model is reproduced by the following equation: 𝑋= 𝑇𝑘𝑃𝑇𝑘 + 𝐸 Where, multivariate factors are presented into the resultant of two new matrices by X  matrix i.e. Tk  and Pk ; Tk  matrix of scores correlates the samples; Pk  matrix of loadings associates the variables, k  is the number of factors available in the model and E demonstrates the matrix of residuals. The trajectories were analysed through R, RStudio and essential codes. The R package ggplot2 was utilized for PCA plots generation. 3. Result 3.1. Investigation of mutation in TSHR  gene All the 21 patients with dysgenesis had mutation in exon 10 among a total of 11 exons in TSHR gene. The mutations we found namely, c.1523C>T (p.Ser508Leu) and c.2181G>C (p.Glu727Asp). Among the 21 patients, only one patient had mutation c.1523C>T (p.Ser508Leu) and 20 patients had the other variant c.2181G>C (p.Glu727Asp). The variants were then analyzed by bioinformatics tools to explore the pathogenic effect. Firstly, the mutations were tested by Polyphen 2, Mutation taster, and PROVEAN bioinformatics tools to see whether they possessed damaging effect or not. We found that the mutation c.1523C>T probably had damaging effect and c.2181G>C variant showed benign effect. Fig 1  represents a chromatogram showing the mutation (c.1523C>T) for the specific participant and Table 2  shows the mutations found in TSHR  gene. 3.2. Effect of mutations on predicted 3D structure of TSHR protein The I-TASSER predicted best structures were designated as wild-type TSHR368-764WT, p.Ser508Leu variant as TSHR368-764 MT1 and p.Glu727Asp variant as TSHR368-764 MT2. Table 3 shows the C-score (Confidence score) for wild-type, MT1 and MT2 was -0.71, -0.61 and -0.63, respectively. The TM score (Template Modelling score) and RMSD were 0.62±0.14, 8.4±4.5Å for TSHR368-764 WT; 0.64±0.13 and 8.2±4.5Å for TSHR368-764 MT1; 0.63±0.13 and 8.2±4.5Å for TSHR368-764 MT2. C-score indicated confidence score to assess the global accuracy of predicted models which is calculated based on the significance of threading template alignments and the convergence parameters of the structure assembly simulations. C-score of higher value [–5,2] suggests a model with a high confidence [ 8 ]. The global quality of the protein model prediction had been assessed by the TM-score. The TM-score represents the similarity between two protein structures and the accuracy of structure modeling. The TM-score (TM-score > 0.5) of the predicted proteins indicated structures were in correct global topology [ 15 ]. From the analysis of TM-score, the target and template (rhodopsin x-ray crystal structure, PDB:1F88) alignments for TSHR368-764 WT, TSHR368-764 MT1, and TSHR368-764 MT2 were 62%, 64%, and 63% respectively. The target-template superimposed structures were displayed in Fig 2A–2C . Later, I-TASSER predicted structure was compared with AlphaFold predicted structure based on analysis of their TM score (0.68) by TM-align algorithm. TM score (0.68) indicated AlphaFold and I-TASSER predicted structures were in same fold with correct global topologies (TM score>0.5). AlphaFold and I-TASSER both provide highly accurate structures [ 8 , 16 , 17 ] while I-TASSER ( https://zhanggroup.org/I-TASSER/ ) got recognition as the No 1 server for protein structure prediction in community-wide CASP experiments. Hence, I-TASSER predicted models were utilized for further analysis. Moreover, the full length TSHR protein predicted by AlphaFold was shown ( Fig 2D ) and the structural alignment of TSHR368-764 for AlphaFold, and I-TASSER was shown in superimposed pose ( Fig 2E ). Fig 3  depicts the structures of the TSHR protein and the small molecules MS437 and MS438. 3.3. Molecular docking and protein-ligand interactions of MS437 and MS438 with TSHR proteins (wild-type and mutant) The structures of the small molecules MS437 and MS438 were optimized. After molecular docking best docking poses for the protein-ligand complexes were selected evalutating their binding affinity and interaction. The binding affinities for the small molecules were obtained from both PyRx software and PRODIGY. The Table 4  showed that the binding affinities of the wild -type TSHR protein (-6 kcal/mol, -5.45 kcal/mol for TSHR368-764WT) were higher compared to the mutant cases (-4.8 kcal/mol, -5.28 kcal/mol for TSHR368-764 MT1 and -5.7 kcal/mol, -5.27 kcal/mol for TSHR368-764 MT2) in both PyRx software and PRODIGY. Also, the binding affinities for the small molecules were obtained from both PyRx software and PRODIGY for AlphaFold predicted structures and the values were included in Table 4 . Total non-covalent interactions were 11, 19 and 12 for wild-type, MT1 and MT2, respectively ( Table 5 ). MS437 bound to Threonine 501 and MS438 bound to Serine 505 and Glutamic acid 506 of transmembrane helix3 (TMH3) in full length TSHR protein [ 6 ] with corresponding amino acid position Thr134, Ser138 and Glu139, respectively in TM-region ( Fig 3 ). We tried to investigate whether these crucial amino acids could interact with small molecule thyrogenic drugs. We found that in case of MS437 none of these three amino acids could interact with both wild-type and mutant cases. On the other hand, MS438 interacted with all the crucial amino acids including Thr134, Ser138 and Glu139 for wild-type case and for the mutant cases (TSHR368-764 MT1 and TSHR368-764 MT2), it could interact only with Ser138. The binding affinities were -7.1 kcal/mol; -5.59 kcal/mol, -5.4 kcal/mol; -5.77 kcal/mol, and -2.6 kcal/mol; -5.50 kcal/mol for TSHR368-764WT, TSHR368-764 MT1, and TSHR368-764 MT2, in both PyRx software and PRODIGY respectively. In Table 6 the binding affinity of wild-type protein was much higher for MS438 than mutants. Also, the binding affinities for the small molecules were obtained from both PyRx software and PRODIGY for AlphaFold predicted structures and the value were included in Table 6 . All the non-covalent interactions ( Table 7 ) were depicted in Figs ​Figs44  and ​and55  for MS437 and MS438 respectively. 3.4. Molecular dynamics simulation MD simulation was performed for each complex of TSHR368-764WT, TSHR368-764 MT1, and TSHR368-764MT2 with two designated drugs (MS437 and MS438) for 50 ns time range. In case of MS437 ( Fig 6 ), the RMSDs for TSHR368-764MT2 (0.973–4.965 Å) displayed less fluctuations for α-carbon atoms than TSHR368-764WT (0.906–5.91 Å), and TSHR368-764 MT1 (0.939–5.504 Å) in Fig 6A . Thus, suggesting that, comparatively MS437-TSHR368-764 MT2 was stable in physiological conditions, while more fluctuations were visible in TSHR368-764WT at 43.8 ns (RMSD 5.91 Å) and TSHR368-764 MT1 till 26.5 ns (RMSD ⁓5.2 Å). The Rg manifested quite similar pattern in TSHR368-764 MT1 and TSHR368-764 MT2. However, TSHR368-764WT exhibited more fluctuations initially up to 5.5 ns and later from 46 ns. The average value remained ⁓25.40 Å for the three protein complexes. However, low compactness in ligand-mutant complexes was observed during simulation ( Fig 6B ). In case of SASA, more deviations were found in TSHR368-764WT (18587.716–24360.945 Å2) compared to TSHR368-764MT1 (18106.599–22314.102 Å2) and TSHR368-764 MT2 (19148.648–22851.2 Å2) complexes. However, TSHR368-764 MT1 and TSHR368-764 MT2 manifested some deviations at 7–19 ns, and 24–43 ns during simulation. Overall, mutant structures TSHR368-764 MT1 and TSHR368-764 MT2 were more stable as MS437 bound complexes than TSHR368-764WT ( Fig 6C ). For MolSA in Fig 6D , TSHR368-764WT showed (21208.043–25437.671 Å2) much fluctuations in the whole run than TSHR368-764MT1 (21135.321–24580.781 Å2) and TSHR368-764 MT2 (21739.906–24397.155Å2). TSHR368-764WT was unstable, but TSHR368-764 MT1 and TSHR368-764 MT2 were more stable as the ligand bound complexes in physiological condition. The RMSF value deviated most for TSHR368-764WT in between 1(368)-30(397) and 326(693)-397(764) residues, where, TSHR368-764 MT1 exhibited more fluctuations in 1(368)-47(415) residues than TSHR368-764 MT2. Overall, TSHR368-764 MT2 was more stable as a complex than others due to least deviation in the whole run ( Fig 6E ). The total number of hydrogen bonds indicated structural rigidity of protein. In case of TSHR368-764WT high frequency of hydrogen bonds (average ⁓633) was observed during interaction while TSHR368-764 MT1 exhibited average 593, and TSHR368-764 MT2 manifested average 600 hydrogen bonds. Among the mutant structures, TSHR368-764 MT2 displayed more structural stability in the simulation ( Fig 6F ). During simulation, for MS438 ( Fig 7 ), the RMSD values of α-carbon atoms remained ⁓5.251 Å in TSHR368-764-WT, ⁓5.53 Å in TSHR368-764 MT1, and ⁓5.39 Å in TSHR368-764 MT2. The fluctuations had been observed in TSHR368-764 MT1 and TSHR368-764 MT2 during 30–50 ns while least was found in TSHR368-764-WT. However, the average RMSD values of all the complexes were almost close ( Fig 7A ). The Rg manifested quite high deviations among three complexes. However, TSHR368-764 MT1 exhibited maximum 27.068 Å, which indicated higher stability than other complexes ( Fig 7B ). The SASA values remained close among three complexes. However, TSHR368-764 WT manifested least deviations during 10–20 ns and 30–50 ns ( Fig 7C ). In case of MolSA, the graphical patterns for three complexes were almost same through the whole MD run ( Fig 7D ). The RMSF value more diverged in TSHR368-764 MT2 till first 15 residues while least deviation was observed for TSHR368-764 WT through whole run. However, three complexes showed quite similar pattern between 130(498)-240(608) residues ( Fig 7E ). The highest number of hydrogen bonds (about 678) was observed for TSHR368-764 MT1 while TSHR368-764 WT exhibited about 669 hydrogen bonds and TSHR368-764 MT2 showed almost 665 to maintain stable conformation. Thus, TSHR368-764MT1 showed highest structural stability among the complexes ( Fig 7F ). Moreover, we had visualized the binding pattern of MS437 and MS438 ligands with the wild-type and mutants TSHR368-764 through the snapshots from MD simulation ( Fig 8 ). In simulation, MS437 exhibited persistent interaction with the residues LEU302(669), ALA306(673), LEU310(677) of TSHR368-764MT2. The MS438 ligand mostly showed stable interactions with the residues LEU100(467), VAL135(502), SER138(505), LEU203(570), PRO204(571), LYS293(660). Both ligands remained within the binding site in stable mutant proteins. 3.5. Principal Component Analysis (PCA) Two PCA models were generated for structural and energy profiles of the protein-ligand complexes to assess and realize the dissimilarities among wild-type and mutant proteins during MD simulation. The scores plot for MS437-protein ( Fig 9A ) and MS438-protein ( Fig 9C ) complexes had exhibited the different clusters for the wild-type and mutants of TSHR368-764. It was observed that in both protein-ligand complexes, TSHR368-764WT and TSHR368-764MT1 were remotely situated. Consequently, pathogenic TSHR368-764 MT1 was liable for the differences. However, TSHR368-764WT and TSHR368-764 MT2 were overlapped. The loading plots ( Fig 9B and 9D ) demonstrated that bond, bond angles, van der Waals energies, and dihedral angles were closely distributed and displayed quite similar graphical pattern. The distribution mainly contributed for PC1 variance while coulomb energy difference contributed to PC2 variance. In MS437-protein complexes, the total 92.7% of the variance had been unveiled by PC1 and PC2, where PC1 expressed 76.1% and PC2 expressed 16.6% of the variance. Moreover, in MS438-protein complexes, the total 88.3% of the variance had been disclosed by PC1 and PC2, where PC1 expressed 72.1% and PC2 expressed 16.3% of the variance. 4. Discussion The newborn screening for endocrine disorders is not frequently practiced in Bangladesh. In this study, we focused on the etiology of dysgenesis types of Congenital Hypothyroid patients having small glands or ectopic gland or agenesis (absent of thyroid gland). Different studies suggested that TSHR  was the major gene responsible for growth and development of thyroid gland [ 2 , 18 ]. TSH binds to the receptor and creates the signaling pathway through G-protein coupled-receptor and Cyclic AMP-mediated adenylate cyclase. The full-length protein structure of TSHR is still under investigation through crystallography. The available structures do not include the all-cytoplasmic residues. Mutations in the TSHR  gene results from loss or gain of function of the protein that causes different phenotypic variations and lead to hyperthyrotopinemia to severe Congenital Hypothyroidism [ 18 , 19 ]. Analysis of TSHR gene showed that two mutations were found, namely c.1523C>T and c.2181G>C in the patients and we analyzed the effect of mutations by using different bioinformatics tools. Almost all the tools such as Polyphen 2, Mutation Taster and PROVEAN were very much popular to analyze the mutational effect. The mutation c.1523C>T was found to be damaging, disease causing or deleterious and c.2181G>C was found to be benign or neutral. Since MS437 and MS438 are thyrogenic potent molecules, we selected these two molecules as ligands for molecular docking with the wild-type and mutant structures of TSHR protein [ 6 ]. The molecular docking analysis showed that the binding affinity for both of the ligands with mutant cases was decreased compared to the wild-type TSHR protein. MD simulation indicated that, the RMSDs for MS437-TSHR368-764 MT2 (average 4.37Å) showed less deviations for α-carbon atoms. Thus, proposing the complex as most stable in biological environments. However, MS438-protein complexes manifested quite close average RMSD values during simulation. The Rg of MS437-TSHR368-764WT exhibited more instabilities at start and end of the simulation. Conversely, TSHR368-764 MT1 and TSHR368-764 MT2 displayed quite similar pattern of lesser compactness as well as more stability for interaction with MS437. In case of MS438, TSHR368-764 MT1 exhibited highest Rg value, which identified higher stability than other complexes. The analysis of SASA and MolSA values revealed that TSHR368-764 MT1 and TSHR368-764 MT2 mutant structures were more stable in their complex form with MS437 than TSHR368-764WT. However, the SASA presented close pattern and MolSA exhibited almost same graphical pattern among the three complexes for the interaction with MS438. In case of hydrogen bonds, MS437-TSHR368-764MT2 manifested average 600 hydrogen bonds which was close to TSHR368-764WT (average ⁓633). The complex was more stable than the other mutant. On the other hand, MS438-TSHR368-764 MT1 displayed maximum structural stability compared to other complexes. Considering RMSF values, MS437 rendered more stability to TSHR368-764MT2 than others. The RMSF value more diverged in TSHR368-764MT2 while minimum deviance was detected for TSHR368-764-WT and TSHR368-764 MT1 mostly remained between both complexes. However, three complexes displayed almost similar stability between 130(498)-240(608) residues while interacting with MS438. Moreover, PCA analysis for MS437-protein and MS438-protein complexes had revealed the existing differences among structural and energy profiles of the structures. It was observable that TSHR368-764 MT1 exhibited much variations than TSHR368-764WT and TSHR368-764 MT2, emphasizing more damaging pattern in TSHR368-764 MT1. In the study, we had utilized allosteric ligands MS437 and MS438 as agonists against the identified mutants for TSHR368-764. These two ligands had ‘drug-likeness’ as well as previously confirmed their efficacy by conducting in vivo  animal studies [ 6 ]. The agonists (MS437 and MS438) displayed different binding sites in the TSHR protein [ 6 ]. After analyzing all data, it can be proposed that low-affinity binding infers, a comparatively high concentration of the ligands can maximally occupy the binding sites to achieve maximum physiological response. Moreover, modifying chemical properties or ligands with novel scaffolds targeting signal-sensitive amino acids surrounding the allosteric binding sites might lead to design agonists with even higher efficiency to activate TSHR [ 19 ]. 5. 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Written by Hopkins Medicine https://www.hopkinsmedicine.org/health/conditions-and-diseases/hashimotos-thyroiditis Original language: English Thyroiditis is when your thyroid gland becomes irritated. Hashimoto's thyroiditis is the most common type of this health problem. It is an autoimmune disease. It occurs when your body makes antibodies that attack the cells in your thyroid. The thyroid then can't make enough of the thyroid hormone. Many people with this problem have an underactive thyroid gland. That's also known as hypothyroidism. They have to take medicine to keep their thyroid hormone levels normal. What is the cause of Hashimoto’s thyroiditis? Hashimoto's thyroiditis is an autoimmune disorder. Normally, your autoimmune system protects your body by attacking bacteria and viruses. But with this disease, your immune system attacks your thyroid gland by mistake. Your thyroid then can't make enough thyroid hormone, so your body can't work as well. Who is at risk for Hashimoto’s thyroiditis? Things that may make it more likely to you for to get Hashimoto’s thyroiditis are: Being a woman. Women are about 7 times more likely to have the disease. Hashimoto's thyroiditis sometimes begins during pregnancy. Middle age. Most cases happen between 40 to 60 years of age. But it has been seen in younger people. Heredity. The disease tends to run in families. But no gene has been found that carries it. Autoimmune diseases. These health problems raise a person’s risk. Some examples are rheumatoid arthritis and type 1 diabetes. Having this type of thyroiditis puts you at higher risk for other autoimmune illnesses. What are the symptoms of Hashimoto's thyroiditis? Each person's symptoms may vary. Symptoms may include: Goiter This is an enlargement of your thyroid gland. It causes a bulge on your neck. It is not cancer. But it can cause problems like pain or trouble with swallowing, breathing, or speaking. Underactive thyroid When your thyroid doesn’t make enough thyroid hormone, it can cause these symptoms: Tiredness Muscle weakness Weight gain Being cold bothers you Depression Hair and skin changes Overactive thyroid When the thyroid is attacked by antibodies, it may at first make more thyroid hormone. This is called Hashitoxicosis. It does not happen to everyone. But it can cause these symptoms: Being hot bothers you Rapid heart rate Sweating Weight loss Tremors Anxiety These symptoms may look like other health problems. Always see your healthcare provider for a diagnosis. How is Hashimoto thyroiditis diagnosed? Your healthcare provider will ask about your medical history and give you a physical exam. You will also have blood tests. These can measure your thyroid hormone levels and check for some antibodies to proteins in the thyroid. How is Hashimoto's thyroiditis treated? Your healthcare provider will figure out the best treatment for you based on: Your age, overall health, and medical history How sick you are How well you handle certain medicines, treatments, or therapies If your condition is expected to get worse Your opinion or preference You will not need treatment if your thyroid hormone levels are normal. But Hashimoto's thyroiditis often looks like an underactive thyroid gland. If so, it can be treated with medicine. The medicine replaces lost thyroid hormone. That should stop your symptoms. It can also ease a goiter if you have one. A goiter can cause problems like pain or trouble swallowing, breathing, or speaking. If these symptoms don't get better, you may need surgery to remove the goiter. When should I call my healthcare provider? Tell your healthcare provider if your symptoms get worse or you have new symptoms. Key points about Hashimoto’s thyroiditis Hashimoto's thyroiditis can cause your thyroid to not make enough thyroid hormone. It is an autoimmune disease. It occurs when your body makes antibodies that attack the cells in your thyroid. Symptoms may include an enlarged thyroid gland (goiter), tiredness, weight gain, and muscle weakness. You don’t need treatment if your thyroid hormone levels are normal. If you have an underactive thyroid, medicine can help. Next steps Tips to help you get the most from a visit to your healthcare provider: Know the reason for your visit and what you want to happen. Before your visit, write down questions you want answered. Bring someone with you to help you ask questions and remember what your provider tells you. At the visit, write down the name of a new diagnosis, and any new medicines, treatments, or tests. Also write down any new instructions your provider gives you. Know why a new medicine or treatment is prescribed, and how it will help you. Also know what the side effects are. Ask if your condition can be treated in other ways. Know why a test or procedure is recommended and what the results could mean. Know what to expect if you do not take the medicine or have the test or procedure. If you have a follow-up appointment, write down the date, time, and purpose for that visit. Know how you can contact your provider if you have questions.

https://www.britannica.com/biography/George-Redmayne-Murray George Redmayne Murray , (born June 20, 1865, Newcastle-upon-Tyne, Northumberland , Eng.—died Sept. 21, 1939, Mobberley, Cheshire), English physician who pioneered in the treatment of endocrine disorders. He was one of the first to use extractions of animal thyroid to relieve myxedema (severe hypothyroidism) in humans. George, the son of a prominent physician, William Murray, received clinical training at University College Hospital, London. He was awarded both his M.B. (1889) and M.D. (1896) by the University of Cambridge . Determined to pursue a career in experimental medicine , Murray in 1891 became pathologist to the Hospital for Sick Children in Newcastle. He also lectured in bacteriology and comparative anatomy at Durham University. From 1893 to 1908 he was Heath professor of comparative pathology at Durham. Appointed to the chair of medicine at Manchester University , he remained there to the end of his career. In 1891 Murray published his most important research, a report in the British Medical Journal on the effectiveness of sheep thyroid extract in treating myxedema in humans. Thyroid deficiency had been recognized as the cause of myxedema in the 1880s, and several researchers had established that an animal could survive the usually fatal effects of thyroidectomy if part of the excised thyroid gland was transplanted to another body location. Sir Victor Horsley, a colleague of Murray’s, later suggested that part of a sheep’s thyroid could be transplanted into human patients to relieve myxedema. Murray surmised, however, that a hypodermic injection of thyroid extract could more effectively be used to correct myxedema in humans, and he was completely successful in his first such attempt at treatment. Subsequent tests substantiated his approach. George Murray was born at Newcastle, the son of William Murray, F.R.C.P. He was educated at Eton and Trinity College, Cambridge, being placed in the first class of the natural sciences tripos of 1886. He qualified in medicine at University College London, in 1888, receiving the Fellowes gold medal in the same year, and completed his training with visits to Berlin and Paris. He acted as house physician in University College Hospital before starting practice in his native city, where he was appointed in 1891 pathologist to the Hospital for Sick Children and lecturer on bacteriology at Durham University. In 1891, too, he made his reputation by being the first to treat myxoedema with thyroid extract given by injection. In 1893 he was made Heath professor of comparative pathology in the University and in 1898 physician to the Royal Victoria Hospital, Newcastle. He relinquished both appointments in 1908 when he was chosen to fill the chair of medicine at Manchester University, which carried with it the office of physician to the Manchester Royal Infirmary. In the 1914—1918 War he served with the 2nd Western General and 57th General Hospitals, and from 1918 to 1919 as consulting physician, with the rank of colonel, to the British forces in Italy. He was a member of the Medical Research Council from 1916 to 1918. He was a contributor to Quain’s Dictionary and Allbutt’s System of Medicine , and at the Royal College of Physicians delivered the Goulstonian Lectures in 1899 and the Bradshaw Lecture in 1905. He resigned his offices at Manchester in 1925, and in retirement lived at Mobberley in Cheshire. Murray was a man of high ability and unassuming friendliness — qualities which effectively dispelled the opposition to his appointment at Manchester in 1908. He married in 1892 Annie, daughter of Edward Robert Bickersteth of Liverpool, and had three sons and one daughter. He died at Mobberley. G H Brown

Written by Nicole Ducharme, DO Endocrinologist Hypothyroidism slows metabolism, which compromises how cholesterol is used by the body. But treatments are available to help your levels get back on track. When someone is affected by hypothyroidism , their metabolism is decreased, including the breakdown of cholesterol and triglycerides. This leads to elevated levels of those fatty substances in the bloodstream, which puts patients at risk of cardiovascular disease. Thyroid replacement therapy reduces LDL (bad cholesterol), total cholesterol, HDL (good cholesterol), and triglycerides. Here’s how. How are Hypothyroidism and Cholesterol Connected? The main role of thyroid hormone is to regulate the body’s metabolism, and therefore it plays an important role in lipid metabolism—the use or storage of fats for energy. If your metabolism is slowed, which is what occurs in hypothyroidism, the breakdown or elimination of cholesterol is also reduced, which leads to higher circulating cholesterol levels in the bloodstream. These elevated circulating levels then put one at increased risk of cardiovascular disease. The correlation between hypothyroidism and abnormal cholesterol metabolism was first reported in 1930.¹ Since that time, there has been a gradual recognition of hypothyroidism and its negative impact on cholesterol metabolism, in particular total cholesterol, LDL (bad cholesterol), and triglycerides. The prevalence of hypothyroidism (marked by elevated TSH and low free T4 ) and subclinical hypothyroidism (marked by elevated TSH and a normal free T4) is 4.3% and 11.1%, respectively, of hyperlipidemic patients.¹ This indicates that both thyroid hormone and TSH play an integral part in the development of abnormally elevated levels of lipids (fats, cholesterol, triglycerides). Can Hypothyroidism Cause High Cholesterol Levels? Yes, hypothyroidism is a secondary cause of high cholesterol levels. The effect of hypothyroidism on lipid metabolism occurs through several different mechanisms due to low T4 and T3 and to the elevation of TSH directly. Firstly, the LDL receptor is protein that sits on the surface of the liver. Its job is to recognize and take up lipoproteins (certain cholesterol particles) and remove them from the bloodstream. Thyroid hormone serves to increase these receptors. However, when thyroid hormone is decreased, as it is in hypothyroidism, the number of LDL receptors on the liver surface and the ability to clear out cholesterol are both decreased, which leads to increased cholesterol. Secondly, the reduction of thyroid hormone also promotes the absorption of cholesterol in the intestine through Niemann-Pick C1-like 1 protein (NPC1L1) and it decreases the breakdown of free fatty acids, which leads to an increase in serum triglycerides.³ In addition, a group of proteins called angiogenin-like proteins (ANGPTL), have been shown to inhibit lipoprotein lipase, an enzyme required to degrade circulating triglycerides.² Thirdly, there is data that having low T3 levels decreases control of sterol regulatory element-binding protein 2 (SREBP-2), which is essential to activate the LDL receptor.³ More recent data suggests that thyroid hormone may play a role in how bile acids are used by the liver. This process (bile acid synthesis) helps to deplete cholesterol stored in the liver and increase the liver’s uptake of cholesterol from blood circulation. When thyroid hormone is not available, this cannot occur.³ Interestingly, serum concentrations of CETP (cholesteryl ester transfer protein), which plays an important role in suppressing HDL (good cholesterol) levels, has been found to be decreased in patients with hypothyroidism. Therefore, HDL is increased in patients with hypothyroidism.⁵ Not only does a decrease in thyroid hormone lead to the above changes, but the elevation in TSH (independent of thyroid hormone) also stimulates cholesterol production, lipolysis (breakdown of triglycerides into fatty acids released from fat cells into the bloodstream), and a decrease in cholesterol clearance. Can Levothyroxine Treatment Help to Reduce Cholesterol in Hypothyroidism? Yes, thyroid replacement has been shown to decrease cholesterol and even normalize cholesterol levels in patients with overt hypothyroidism. It is not recommended to start treatment in those older than 65 with TSH levels below 10 mlU/L because of the risk of increased mortality with thyroid treatment in this patient population.² In addition, all patients diagnosed with high cholesterol should have a screening of TSH and free T4 levels before starting treatment. Can Dietary Changes Reduce High Cholesterol in Hypothyroidism? The cornerstone of high cholesterol treatment is diet and exercise. However, in patients with secondary hyperlipidemia, such as those with hypothyroidism, diet alone is not likely to significantly decrease cholesterol levels until the underlying disorder is treated. Therefore, getting the thyroid level into optimal range in those with hypothyroidism is essential to getting cholesterol levels optimally treated. References: Hixing Liu, Daoquan Peng. Update on dyslipidemia in hypothyroidism: the mechanism of dyslipidemia in hypothyroidism. Endocrine Connections . 2022, 11 :e210002 Xin Su, Hua Peng, Xiang Chen, Xijie Wu, Bin Wang. Hyperlipidemia and hypothyroidism. Clinica Chimica Acta . 2022, 527 :61-70 Leonidas H. Duntas, Gabriela Brenta. Thyroid hormones: a potential ally to LDL-cholesterol-lowering agents. Hormones . 2016, 15(4) :500-510 Angeliki Stamatouli, Pablo Bedoya and Sahzene Yavuz. Hypothyroidism: Cardiovascular Endpoints of Thyroid Hormone Replacement. Frontiers in Endocrinology . 2020, 10 :1-8 Xin Su, Xiang Chen, Hua Peng, Jingjin Song, Bin Wang, Xijie Wu. Novel insights into the pathological development of dyslipidemia in patients with hypothyroidism. Bosn J Basic Med Sci . 2022, 22(3) :326-339

Written by Thyroid Patient Advocacy One of the early methods of treating an under active thyroid was to fry sheep’s thyroid glands and eat them with currant jelly or brandy. This was considered preferable to the previous treatment of grafting sheep’s glands beneath the skin of thyroid patients or injecting thyroid extract (1). In 1894, E. Merck, introduced one of the first thyroid preparations in the world (Triiodothyronine siccatum) (2). According to Burgess a woman in the US was first treated for hypothyroidism with thyroid extract in 1896. She began taking it when she was 39 years old, and continued treatment for the rest of her life until she died at age 91(3). Natural desiccated porcine thyroid extract contains Thyroxine (T4) and Triiodothyronine (T3) hormones, as well as T2, T1 and calcitonin. T2 is considered necessary for production of the deiodinase enzyme that helps convert T4 into T3. T3 is the active hormone that regulates the metabolism and is of short duration in the body. T4 has a much longer half-life in the body. T1s physiological role was still being evaluated until very recently when it was found that T1 has an influence on the electrical input and charge of the brain and various mental disorders, including multiple sclerosis and Lou Gehrigs syndrome, which can be a result of not enough T1 to recharge the brain. (4) New findings research suggests that although the compound, known as T1 amine, is a derivative of thyroxine, an essential thyroid hormone that influences development, body temperature, metabolic rate and cardiac performance, it has the opposite effect of thyroxine. (5). This research also suggests that T1 amine affects several organ systems. Consequently, if its molecular and cellular actions can be precisely described, physicians will be in a better position to treat a variety of cardiovascular and endocrine diseases, as well as mental health disorders. said David Grandy, Ph.D., Associate Professor of Physiology and Pharmacology, and Cell and Developmental Biology in the OHSU School of Medicine. “here we thought we knew thyroid hormone so well, only to find out there’s this whole new aspect of it,” said Grandy, co-author of a study published in an online edition of the journal Nature Medicine. T1 amine’s normal function in the body may be to counteract, or keep in check, thyroid hormone’s actions.” (6) For almost 60 years, desiccated porcine thyroid was the only thyroid treatment and was used successfully from 1894 until 1958, when synthetic T4 only medications first appeared on the market. Since then, doctors have increasingly viewed desiccated thyroid as old fashioned and obsolete. Unfortunately, teaching about the use of natural thyroid extract in medical schools stopped suddenly in 1975, and many doctors are untrained in its use. Many patients on Internet support groups for hypothyroid patients have reported that their doctors have never even heard of desiccated thyroid. Doctors obtain their information about drugs mainly from universities and from pharmaceutical companies. When a medical treatment with a long history of success is relegated in favour of one that sometimes works, but is overall less effective and is more expensive, doctors and patients have a serious problem. Around about the same time that natural desiccated thyroid was relegated in favour of synthetic preparations, there was a similar argument made for infant formula over breast milk. Infant formula was marketed as being superior to natural mother’s milk, and mothers who nursed their babies instead of feeding them formula were seen as perhaps less well informed. That situation has turned around completely since then. However, the belief that synthetic T4 is superior to natural desiccated thyroid, which contains all the thyroid hormones, still exists among endocrinologists and general practitioners. The question arises about how these doctors got the impression that desiccated thyroid is unstable. The following quote from the bible of thyroid treatment, Goodman and Gilmans The Pharmacological Basis of Therapeutics, provides some light on this question. “Several years ago (1963) a large batch of material came into the hands of a number of distributors in the United States and Europe and, although of proper iodine content, it later proved not to be thyroid extract at all. This episode gave desiccated thyroid a bad name because several publications about the unreliability of thyroid extract appeared before the hoax was uncovered” The best-known brand name of desiccated thyroid is Armour Thyroid, manufactured by Forest Pharmaceuticals Inc. in the US. Armour Thyroid and several other thyroid medications were grand fathered in when Congress passed the Kefauver-Harris Drug Efficacy Amendments of 1962, to tighten control over drugs. Before marketing a drug, firms had to prove safety and effectiveness for the products intended use. The requirement was applied retroactively to 1938, when the FDC Act was passed. Pre-1938 drugs were allowed because they were generally recognised as safe and effective, provided no evidence to the contrary developed. Too much evidence to the contrary developed concerning the levothyroxine products and the FDA decided none of them was generally recognised as safe and effective, so the levothyroxine products lost their grand fathered privilege and had to go through the NDA process. Armour thyroid retains its grand fathered status since no evidence to the contrary has developed concerning its safe and effective status. Until around 1970, the hormone concentrations of thyroid medications were titrated based on iodine content, but since then, they have all (including desiccated thyroid) been assayed by their hormone content. However, desiccated thyroid has been assayed the same way as other thyroid hormone medications for several decades (7) and it is recalled far less often for stability issues than its synthetic counterparts are. For details, see a 2003 newsgroup post that compares FDA recalls of different thyroid medications (8). Dr Raymond Peat states, When synthetic T4 first became available, and in healthy young men it acted “like the thyroid hormone”, older practitioners recognized that it was not metabolically the same as the traditional thyroid substance, especially for women and seriously hypothyroid patients, but marketing, and its influence on medical education, led to the false idea that the standard Armour Thyroid USP wasn’t properly standardized, and that certain thyroxine products were, despite the fact that both of these ideas were shown to be false” (9) When NHS medical practitioners are asked by patients why they are not prescribing Armour Thyroid for patients who are unable to tolerate T4 (Levothyroxine) typical responses are: 1. Armour Thyroid was found to have had potency problems in the past 2. Armour Thyroid is an unreliable preparation in terms of active thyroid. 3. There is no adequate quality control. 4. The Medicines Control Committee will not allow this product to be licensed because of previous potency problems. 5. Thyroxine is the only medication which provides steady hormone levels. 6. Because of issues surrounding “mad cow disease” and other ailments, some are reluctant to offer animal based therapy to patients when a safe, effective well-studied synthetic preparation is widely available. As determined by Armour Pharmaceutical Company and other participating laboratories, the liothyronine and levothyroxine content in Armour thyroid is well within the specifications set by the U.S. Pharmacopoeia. The precision of the assay procedure as determined by Armour, Eli Lilly, and the FDA is considerably better than that reported by Pharmaceutical Basics (10). What many general practitioners fail to recognise is that the thyroid powder that contains the active ingredients for Armour Thyroid is derived from the thyroid glands of ‘grain-fed’ pigs and hogs. The USDA changed the requirements about feeding ground animal remains to pigs several years ago. The porcine thyroid glands that go into Armour Thyroid tablets are only derived from animals that do go into the US food supply. If any animal were rejected from the food supply, its thyroid gland would never get into Forest Pharmaceuticals product. Armour Thyroid has been on the U.S. pharmaceutical market for over 100 years and there are no reports of any Armour Thyroid patient ever contracting CJD”. As some endocrinologists imply, T4 replacement (and T4/T3 replacement, which they discourage) will indeed work well for some hypothyroid patients. For others, however, synthetic replacement therapies are clearly ineffective. Armour is highly effective for many hypothyroid patients, who often have lingering symptoms on synthetic medication. It is absorbed more completely and more consistently than pure T4 because all the hormones are protein bound, which is the natural way for the hormones to be absorbed. In the natural, desiccated form, T3 appears to spread out its effects more evenly, causing fewer of the ups and downs that patients often experience with synthetic T3. For many patients, taking desiccated thyroid instead of T4 alone or even a T4/T3 combination relieves many symptoms that were not reversed previously Armour Thyroid does not have the stability problems those synthetic T4 medications are known for. It is remarkably uniform and well absorbed and the potency is sufficiently standard that variation cannot be detected clinically (11). It is a highly satisfactory preparation for clinical use and for most purposes; desiccated thyroid is to be preferred to any other type of thyroid preparation for many people (12). A 2001 study that apparently received little notice proves what thyroid patients have been saying for years desiccated thyroid is more effective in relieving hypothyroidism symptoms than the synthetic hormones (13). Today, The Endocrine Society’s membership consists of over 11,000 scientists, physicians, educators, nurses and students, in more than 80 countries. As this Society has previously undertaken trials into synthetic T4 v T3, it should not be impossible for them to conduct a large, multi-centre, double blind, randomised, controlled trial to prove the efficacy or otherwise of T4 v T4/T3 combination v natural desiccated porcine thyroid extract (Armour Thyroid). Patients suffering from an under active thyroid living in the UK who have benefited from switching from synthetic to natural thyroid extract (Armour) would undoubtedly be astonished and dismayed to read the Endocrine Societys trial report which indicated once again that synthetic T4 alone provides the only answer to this condition (14). References: George R. Murray, M.D., D.C.L., F.R.C.P., The Life History Of The First Case Of Myxoedema Treated By Thyroid Extract British Medical Journal; March 13 1920:359-360. Merck Thyroid History http://www.thyrolink.com/service/history.htm Burgess AM. Myxoedema, Controlled by Thyroid Extract for 52 years: Case. Ann Int Med 1946;25:146-150 D. A. Versendaal and Dawn Versendaal-Hoezee “Contact Reflex Analysis” Hoezee Marketing, 1993, p. 33. 3-Iodothyronamine is an endogenous and rapid-acting derivative of thyroid hormone Katherine Suchland, Paul Kruzich, Dane Crossley II and James Bunzow, Department of Physiology and Pharmacology, OHSU; Matthew Hart, Department of Pharmaceutical Chemistry and Cellular & Molecular Pharmacology, UCSF; Grazia Chiellini, Sabina Frascarelli, Simonetta Ronca-Testoni, Riccardo Zucchi, Dipartimento di Scienze dell’Uomo e dell’Ambiente, Sezione di Biochimica, Universita di Pisa, Italy; Yong Huang and Emil Lin, Department of Biopharmaceutical Sciences, UCSF; and Daniel Hatton, Department of Behavioral Neuroscience, OHSU. Oregon Health & Science University, the University of California, San Francisco (UCSF) and Universita di Pisa, Italy. Nature Medicine 10, 638 642 (2004) Published online: 16 May 2004,http://www.nature.com/nm/journal/v10/n6/abs/nm1051.html Blumberg KR, Mayer WJ, Parikh DK, Schnell LA: Liothyronine and levothyroxine in Armour thyroid. J Pharm Sci 1993; 76: 346. FDA Enforcement Report Index http://www.fda.gov/opacom/Enforce.html Letter from Raymond Peat,Independent Research, Eugene,Oregon. http://bmj.bmjjournals.com/cgi/eletters/314/7088/1175 Basier VW, Hertoghe J, and Eeekhaut W. Thyroid Insufficiency. Is Thyroxine the Only Valuable Drug? J Nutr Environ Med 2001;11:159-166. Determination of Liothyronine and Levothyroxine in Thyroid Preparations by Liquid Chromatography: Steven L. Richheimer and Charlotte B. Jensen. Received February 14, 1985, from the Quality Control Laboratory, Pharmaceutical Basics, Inc., Denver, CO 80223. Accepted for publication November 14, 1985 Gilman AG. Page 1479 in: Goodman and Gilman’s the Pharmacological Basis of Therapeutics. 4th ed. The Macmillan Company, 1970. Salter WT: Symposium on Hormones. New Engl J Med 1941;709 Basier VW, Hertoghe J, and Eeekhaut W. Thyroid Insufficiency. Is Thyroxine the Only Valuable Drug? J Nutr Environ Med 2001;11:159-166. (Sawka.A. et.al.Does a Combination Regimen of Thyroxine (T4) and 3,5,3Triiodothyronine Improve Depressive Symptoms Better Than T4 Alone in Patients with Hypothyroidism? Results of a Double-Blind, Randomized, Controlled Trial, Journal of Clinical Endocrinology and Metabolism, JCEM 2003 88: 4551-4555)

Written by Ron Kennedy Smothermon, M.D. Dr. Barnes’ Epidemiological Studies Dr. Barnes did an extensive analysis of the autopsy records of people who died in Graz, Austria in the years 1930 and 1970, plus selected years in between — around 70,000 cases in all. Graz is a city with a stable population of around 230,000 people. There is only one hospital, the National Hospital (Landskrankenhaus), and by a two-hundred-year-old royal decree, everyone who dies in the National Hospital must have an autopsy. About seventy percent of deaths in Graz occur in the National Hospital. Dr. Barnes noticed that during World War II, the incidence of death from coronary artery disease went almost to zero. After the war, that incidence went back to prewar levels. He believes people who would have died from heart attacks later in life died prematurely during the war — primarily from infectious diseases, because antibiotics were generally unavailable during that time. After the return of availability of antibiotics, the same people who would have died of infectious disease again began to die of coronary artery disease. In the cases from the year 1930, the great majority of deaths in Graz were caused by tuberculosis. Tuberculosis kills its victims at the average age of forty (recall Wolfgang Amadeus Mozart — dead at age forty from tuberculosis). Dr. Barnes is one of those people who thinks for himself. He looked at the evidence, as follows: prior to the era of antibiotics, coronary artery disease was almost unknown. A certain segment of the population was susceptible to tuberculosis and infections in general, and they died young from infectious disease, primarily tuberculosis. Then came antibiotics and an entirely new population of people appeared: people who were susceptible to both infection and to coronary artery disease; except now they were enabled to live long enough to die from the slower of the two diseases: coronary artery disease. Coronary artery disease kills its victims at the average age of 66, 26 years later than the previous big killer, tuberculosis. Dr. Barnes noticed that the death rate from coronary artery disease in 1970 was ten times that of 1930. Statistically, the death rate would be expected to be double, not ten times the 1930 rate. The only way he could explain this to himself was that the people who were saved from tuberculosis were almost all dying of coronary artery disease, and the people who did not need saving from tuberculosis were not susceptible to coronary artery disease either. They would die at an older age of something else. Dr. Barnes combined this insight with his vast clinical experience of thousands of people treated with thyroid replacement therapy and realized that the incidence of both infection and coronary artery disease is dramatically reduced by thyroid replacement. This led to the realization that thyroid deficiency is the common denominator in both susceptibility to infection and coronary artery disease. In other words, there is a segment of the population (about forty percent according to Dr. Barnes) which accounted for almost all deaths from infectious disease before the invention of antibiotics and is now destined to account for almost all of the mortality statistics related to heart attacks, and these same people are hypothyroid. Dr. Barnes’ advice to you is to take your morning basal temperature, and determine if you are one of those people and, if you are, find a doctor who will prescribe thyroid replacement therapy for you. Ah, but there is the rub, for while the incidence of hypothyroidism was well appreciated before the invention of blood lab tests for the disease, now most doctors think it is rare. Remember, almost all doctors attend The Church of the Holy Lab Test on a regular basis. If the clinical picture presented by the patient conflicts with the Holy Lab Test, the clinical picture is ignored. According to this point of view, you cannot be hypothyroid unless you have a low T3 and/or T4 blood level. So, if you have hypothyroidism, unless you have the proper lab result, you will have to become a discriminating consumer and conduct a search for a qualified physician. If I were you, I would call up armed with knowledge, and boldly ask to speak to the doctor before making an appointment. Ask the right questions. How common is hypothyroidism? Do you know about the taking of basal temperature? How reliable are lab tests in diagnosing hypothyroidism? Do you know of the work of Dr. Broda O. Barnes? Get the right answers or do not waste your time and money with that doctor. If you think about it, Dr. Barnes’ ideas dovetail nicely with what we now know about the mechanism of coronary, nay any, artery disease. We now believe this to be a free radical disease. Excess hydroxyl radicals left over from lipid peroxidation cause tiny areas of inflammation, then necrosis (tissue death), in the walls of arteries. When the body tries to repair this necrosis it forms a scar tissue and incorporates into the scar tissue calcium and cholesterol. The body’s natural defense to this process is to produce antioxidants, which neutralize hydroxyl free radicals before they can do damage. Thyroid hormones regulate the rate of metabolism and, when in inadequate supply, the rate at which antioxidants are produced would, of course, be slower. Therefore, there would be less antioxidants around to do the job and atherosclerosis would proceed more rapidly. The sequence is clear. Low thyroid function _ decreased antioxidant production _ vascular lesions _ scar (plaque) formation _ eventual artery blockage _ heart attack or _ stroke ( «brain attack,» as it is now called), or _ gangrene, or _ any set of symptoms caused, or contributed to, by decreased blood flow to organs. It also makes sense that low thyroid function would make one susceptible to infection. The entire chemical factory we know as the immune system, is running only as fast as the amount of thyroid hormone which penetrates the immune system cells. Therefore, low thyroid _ depressed and slowed immunity _ increased likelihood of infection. It all fits together so well! Arthritis Hypothyroidism predisposes a person to arthritis, and thyroid replacement therapy often brings arthritic symptoms under control. In severe cases of arthritis it may be necessary to add a small dose of prednisone — from five mg. every other day up to five mg. twice daily. (I prefer natural hormones for hormone therapy, but this may be the one place prednisone, a synthetic hormone, should be used — only in low doses and only because the effect of cortisone treatment in arthritis wears off after a few days.) Some of the ill side effects of prednisone are due to its thyroid-suppressing effect and can be avoided, at least at low doses of prednisone, by supplementing thyroid hormone to the level required to maintain the basal temperature between 97.8 and 98.2. Where gouty arthritis is concerned, thyroid replacement also helps here. Gout is caused by an inability to metabolize uric acid and the accumulation of uric acid, notably in the drainage system of the kidneys (as stones) and in the joints, especially the big toe. If the basal temperature (and thus the basal metabolic rate) is low, naturally the body is even less able to metabolize uric acid. Although thyroid replacement is not specific to gout, it is a valuable adjunct to the treatment of this painful disease. Again the problem is that doctors rely on unreliable laboratory tests to determine the presence or absence of need for thyroid replacement therapy, when the only reliable test is the basal temperature. Diabetes and Hypothyroidism The symptoms of diabetes are related to the poor control of blood sugar present in this disease due to either inadequate insulin production from the pancreas or a resistance in the cells of the body to the effect of insulin. It is as if, in some people, the body becomes immune to the effects of insulin. Before the discovery of insulin, the diagnosis of diabetes was a death sentence. The average person lived less than five years after diagnosis, and the usual cause of death was tuberculosis. One could say that part of being a diabetic was a weakness toward contracting and dying from tuberculosis. Insulin was isolated by Canadian doctors Banting and Best in 1922, and for a few years the medical world was optimistic that diabetes would be cured. What happened instead was that controlling blood sugar with insulin allowed diabetics to live longer lives. This revealed aspects of diabetes, which previously had been no problem, because the diabetic died before they could appear. Diabetics were observed to develop atherosclerosis far earlier than non-diabetics, and this became the major killer of diabetics later in life, by heart attack or stroke. However, they die at a much younger average age than do other sufferers of atherosclerosis. It has been discovered that even before the onset of diabetes, the diabetic-to-be is developing atherosclerosis at an accelerated rate. A weakness to tuberculosis and early atherosclerosis: just like hypothyroidism! And, indeed, if you check the diabetic for a low basal temperature, it often turns out that hypothyroidism is present. It has long been known that the classical test for diabetes, the GTT or glucose tolerance test, cannot distinguish between diabetes and hypothyroidism. However, sometimes doctors forget this and fail to collect the basal temperature (or worse yet rely on the T3/T4 tests) and thus treat hypothyroid patients for diabetes, which they do not have! Also, many true diabetics also have hypothyroidism, which is overlooked and not treated for the same reasons. The complications of diabetes, such as cataracts, heart disease (and atherosclerosis in general) and kidney disease are not present in the diabetic, if that diabetic is producing plenty of thyroid hormone. These complications also can be prevented in the diabetic patient who also is hypothyroid, simply by adding thyroid replacement therapy. The point is that whenever diabetes is suspected, it should always be distinguished from hypothyroidism. This can be done by testing not only glucose tolerance but also insulin tolerance. A GTT should never be ordered alone. Second, even if a person is correctly diagnosed with diabetes, hypothyroidism should always be suspected anyway, and a basal temperature should be done with thyroid replacement, if indicated by a low reading. Many «prediabetics» are actually undiagnosed hypothyroid cases and, if not recognized as such, the opportunity to treat with thyroid and thus prevent atherosclerosis will be lost. Lung Cancer and Emphysema It appears from the extensive epidemiologic studies conducted by Broda Barnes on deaths occurring in Graz, Austria, that here are two other diseases to which there is a shared susceptibility, along with the susceptibility to tuberculosis, and those are lung cancer and emphysema. His data indicate that people who have tuberculosis are twenty times more likely also to have lung cancer than the average person. As regards emphysema, part of the cause of this diseas is chronic bronchitis, i.e., repeated infections of the bronchi. People who finally end up with emphysema are people who smoke and/or have had multiple infections of their breathing tubes. Infections produce coughing in the presence of obstruction of the airways by mucus. Coughing into obstructed lung tubes causes the pressure in those tubes to back up into the alveoli, the tiny sacs in the lungs where oxygen and carbon dioxide are exchanged. When the walls of these sacs rupture under pressure from coughing and scar tissue forms this is called «emphysema.» As we already have seen, people who are especially susceptible to infections are more likely to be hypothyroid, and the addition of thyroid replacement therapy raises resistance to infection to normal levels. This protects against bronchitis and therefore against the eventual development of emphysema. Sources: Barnes BO, Barnes CW Heart attack rareness in thyroid treated patients. Charles C. Thomas; Springfield, IL 1972 Barnes BO, Galton L Hypothyroidism: the unsuspected illness. Harper & Row Publishers; New York, NY 1976 Barnes BO Headache – etiology and treatment. Federation proceeding 1947; 6:73 Barnes BO Etiology and treatment of lowered resistance to upper respiratory infections. Federation Proceedings 1942;69:808 Barnes BO The treatment of menstrual disorders in general practice. Arizona Medicine 1949;6:33 Barnes BO, Ratzenhofer M One factor in increase in bronchial carcinoma. JAMA 1960;174:2229

Written by Datis Kharrazian, PhD, DHSc, DC When you develop Hashimoto’s you lose something called “immune tolerance” and your immune system starts attacking its own tissues. The thyroid is very vulnerable to autoimmune attacks because it has no antioxidant protection. Thyroid tissue also contains many proteins structurally similar to foods that commonly trigger immune reactions. For instance, some amino acid sequences in thyroid tissue are identical to amino acid sequences in gluten. If you are unknowingly gluten sensitive or have celiac disease, you have the potential to trigger an autoimmune attack against your thyroid every time you eat gluten. This is called cross-reactivity and it happens with various foods and body tissues, including the brain. Many people with Hashimoto’s are genetically predisposed to gluten sensitivity or celiac disease.

Written by Datis Kharrazian, PhD, DHSc, DC, MS, MMSc, FACN Listen to the podcast on iTunes . (Also available on major podcast platforms). Watch the YouTube video . Not all thyroid supplements are equally effective – in fact, some can actually make your hypothyroid symptoms worse. Before we go over the best and worst supplements for your thyroid, you first need to understand some of the underlying mechanisms of hypothyroidism. Around 95-98 percent of cases are caused by an autoimmune disease called Hashimoto’s, which determines which supplements will work best for those patients. You might have come across the mistaken belief that hypothyroidism is caused by a nutritional deficiency of iodine, tyrosine, or thyroid glandulars. This is an outdated theory from the 1950s and ‘60s that unfortunately still persists today. In actuality, iodine and tyrosine are not the best options for those with Hashimoto’s – and thyroid glandulars should never be used as a replacement for thyroid medication. The worst supplements for Hashimoto’s hypothyroidism Iodine My stance on iodine was controversial when I included it in my first book, Why Do I Still Have Thyroid Symptoms? But since 2010, research has continued to prove that Hashimoto’s patients should minimize their iodine consumption. Your thyroid gland only needs about a pinhead of iodine over the course of many months, which is easily met through everyday foods. The idea that hypothyroidism is caused by a lack of iodine is not only false – the supplement can actually make thyroid symptoms worse. When you have Hashimoto’s, your immune system targets and attacks thyroid peroxidase (TPO) enzymes in the thyroid gland. The common misconception is that iodine benefits hypothyroid patients because it stimulates TPO activity, but numerous studies have shown that the stimulating effect of iodine on TPO actually increases autoimmune attacks. In fact, epidemiological studies show a dramatic increase in hypothyroidism in areas of the world where iodized salt is introduced. Studies also show many with Hashimoto’s hypothyroidism experience dramatic improvement of symptoms when they begin an iodine-restricted diet. To learn more about iodine and hypothyroidism, please check out this interview . Tyrosine Tyrosine is an amino acid and a building block for thyroid hormones. Like iodine, many people still follow the outdated belief that tyrosine supplements support thyroid function. While no research has shown that it increases thyroid hormones, studies have shown that it promotes catecholamines. These adrenal stress hormones raise energy in a similar way to caffeine. Thyroid glandulars The most common misconception about thyroid glandulars is that they can substitute for thyroid medication, which they can’t. No quality research has been done on the impact of thyroid glandulars on thyroid function, but some patients report feeling more energy even though their levels of thyroid hormones remain unchanged. So while you should never replace your prescribed thyroid medication with thyroid glandulars, you might notice some benefits from adding them to your daily regimen. The best supplements for Hashimoto’s hypothyroidism Now that we have addressed three controversial supplements, let’s go over the best supplements that have been proven to help support thyroid health when you have Hashimoto’s hypothyroidism. Vitamin D Good for general immunity, bone health, and brain health, vitamin D is a beneficial supplement for most of the population – especially since modern diets fail to provide a sufficient amount. It’s even more beneficial for those with Hashimoto’s hypothyroidism. Numerous studies show vitamin D has an immune-modulating role, which is critical for people with these conditions. Some studies also show that vitamin D positively impacts thyroid inflammatory responses. Magnesium Magnesium is a safe supplement that many use to help with sleep and stress. Some research shows that magnesium calms thyroid inflammation and could be beneficial for those with thyroid conditions. Selenium Selenium dampens the inflammatory response and TPO activity. Myo-inositol Myo-inositol is a B vitamin variant. Some studies show taking selenium and myo-inositol together reduces the thyroid autoimmune response. Antioxidants Antioxidants such as glutathione, turmeric, resveratrol, apigenin, and rosemary extract have anti-inflammatory properties that are useful for thyroid swelling, inflammation, and autoimmunity. In addition to a supplement, you can also incorporate more antioxidant-rich foods into your diet. Supplements can’t reverse an inflammatory diet and lifestyle While supplements can help support your thyroid health, the most important strategy to manage Hashimoto’s hypothyroidism is to customize a diet and lifestyle approach that minimizes exposure to inflammatory triggers while improving immune resilience . I teach evidence-based strategies to help patients manage their Hashimoto’s in my course, Hashimoto’s: Solving the Puzzle. Together, we go over how to use diet, nutrition, lifestyle, and nutraceutical approaches to personalize a plan based on your unique needs.

Written by Dr Bjørn Johan Øverbye Why hypothyreose symptoms, when blood-tests are normal? According to a growing number of doctors there are at least two major categories of patients needing thyroid hormones: Hypothyroidism Type I and Type II. Type I is the classical problem: low thyroidhormon in the blood,Type II is heterogenic group where thyroid hormone tests are normal but the function of the hormones on the cells are sadly failing. These are the patients who have “normal tests”, suffer from the symptoms of lack of hormones and get better from thyroid hormone therapy. Hypothyroidism from the mainstream into the light! The historical proof A major problem today is the doctor’s reliance on lab -test’s instead of trusting their eyes, ears, hands and brains. Hypothyreose, in earlier days of medicine called myxedema, is largely a clinical diagnose. Ever since the first description of myxedema and its identification as caused by an injured thyroid gland as early as 1873, the diagnose has been done by clinical investigation. The great thyroid expert of past generation professor Broda Otto Barnes in USA relied nearly 100% on clinical examination plus taking the morning temperature. In his now classic book Hypothyroidism; The unsuspected Illness[i] from 1976 he could look back on 40 years of successful scientific research and clinical work. His conclusions rests on the meeting with thousands of myxedemic patients, today called hypothyreosis or even better called: hypometabolic patients due to thyroid hormone failure. Thyroid problem a part of the greater picture of energy-failure Before going further I should mention the new trends emerging mainly from clinical and theoretical work by doctor John C Lowe [ii] also USA. Low followed in the footsteps of Barnes but with access to a far more advanced science he could explain in detail what Barnes could only experience in daily practice when giving thyroid hormones to patients with low basal temperature plus symptoms of clinical hypothyreose. Having the privilege to be given inspiration and personal advices b Lowe in my own clinical work here in Norway I came to appreciate the importance of believing the patients more than the lab. I also came to understand even more by investigating in depth more than 2000 patients that what we see is this: Myxedema later called hypothyroidism is a manifestation in a decay in energy-production in the cells of the body due to lack of thyroid-hormone effects on the cells. The energy function puts things straight Failure to produce biological energy(E) [iii] results in a proportional decay in metabolism which is the name we use for a cells ability to do work (W). Doing work cells also produce heat, measured as temperature. Since the universal law of energy is: Energy = Work + Temperature [iv] We see that less energy leads to less Work and a colder body, precisely as Broda Barnes described. Ability to do work is called metabolism, so having less energy available leads to hypo-metabolism. Since thyroid-hormones makes cells produce energy faster from free fatty acids, glucose, Oxygen, water and nutrients, more thyroid hormones are increasing Energy and accordingly metabolism. When thyroid hormone effect falls we enter a hypo-metabolic state which is typical for lack of energy as that which goes with less thyroid hormone effects. Therefore hypothyreose leads to hypo- metabolism; but metabolism is not always cased by hypothyroidism, if we bay that means less available thyroidhormones due to a thyroid gland defect. Hypometabolism more than just low thyroid hormones in blood As a matter of fact there are many causes for hypometabolism, but interesting enough the majority leads to a reduced effect of thyroidhormones, therefore the majority of hypometabolic states are also in a way hypothyroidism; but with the exception that apart from pure/classical hypothyroidism, the majority of other states have enough thyroid hormones. But because other factors such a depleted adrenal glands, estrogen dominance, use of anabolic hormones, malnutrition and so on leads to a weakening of thyroid hormones on the cells; one can if ignoring the cause, improve patients health with thyroid hormone supplements. This is of course not attacking the problem, but it is a temporary relief. This is what professor Broda Barnes observed in a period when lab tests were sparse; that as much as 40% of all hypometabolic patients improved their health by using thyroid hormones. Later research proved that 2-20% of all hypometabolic patients had thyroid hormone deficiency (depending on population, gender, age, nutrition etc.) [v] This caused a schisma that doctor Lowe tried to solve 30 years after Broda Barnes period by introducing the concept of Thyroid Hormone Resistance (THR): enough hormone in the blood but unresponsive cells. The situation could be fixed with addition of extra thyroid hormones. Hypothyreose type I and II We thus have the following clinical situation 1. Clinical picture identical to hypothyreose + Lab tests show low thyroid hormones in blood + Gets better from thyroid hormone therapy = Hypothyreosis I 2. Clinical picture identical to hypothyreose + Lab tests show normal thyroid hormones in blood + Other tests not normal + Gets better from thyroid hormone therapy = Hypothyreosis II What are the other lab tests that are abnormal? Having tested more than 2000 hypometabolic patients we today starts to get a fair knowledge of the terrain. It is complicated and very intricate and demands a long experience to understand and convert into practice. It will be dealt with later on. As for now, remember: Hypometabolism is the name for the underlying phenomena appearing as hypothyroidism or hypothyroid-like states. Of these as previously mentioned approximately 2-20% are Hypothyroidism I (depending on age, nutrition, medication of the population investigated.), the remaining 20-38% are Hypothyroidism II. Both will be relived by thyroid hormone therapy, but Hypothyroidism II should definitively be treated by removing what blocks the thyroid hormone effect by all means. In many cases this is possible and making it unnecessary to use thyroid hormone therapy; but when the ideal goal is not reached some thyroid hormones has to be added. Out of the stagnant pool of industrial medicine Revolution means evolving again, anew. Hypothyroidism has for years been a stagnant pool if we are to trust the stories from more than 2000 patients I myself have personally in depth interviewed and the reports written by brave pioneers like professor Broda Otto Barnes and doctor John C Lowe. This stagnation is not due to lack of intelligence or integrity of the generation we could call the “industrial generation of doctors” when medicine turned away from clinical work to the current New Public Management Medicine where there is a high reliance on labwork; without necessary skepticism to its usefulness. To befree oneself from the stagnant pool and doing justice to the genius of ones own creative abilities one has to look at the situation from a different angel: the clinical situation[vi]. Treating patients the doctor of the new area questions the sick, uses his/her eyes and ears and hands to collect data. The doctor then collects labdata from a variety of body parameters (more than 50 oftentimes) including body-temperature measurements done by patients at home and then the doctor adds all the factors together and finally settles on thyroid-hormone therapy or not plus other chemicals needed to correct the situation. When going through such a process a doctors archive will eventually over the years contain two groups of patients if he sorts the sick according to one parameter: patients getting better from thyroid hormone or not. Looking back the doctor will get a very simple definition of his/her patients: People needing thyroid therapy to get better or healed, and those for whom thyroid therapy has no effects. So instead of the tedious feuds in the stagnant pool we simply end up with a new and fruitful situation: Sicknesses that can be improved by thyroid therapy or not. So we now ask: Who will improve their quality of life using thyroid therapy? This is nothing different than asking: Who benefits from cortisone therapy? This also befree us from the problem of what type of product and how much is best. We simply look to the clinical results and observe how labdata change as we approach and finally reach the correct dose. Sources: [i] Barnes, Broda Otto (1976). Hypothyroidism: The Unsuspected Illness. HarperCollins. ISBN 0-690-01029-X. [ii] Lowe John C : · McDowell Publishing Company; 1st Edition (Februar 1, 2000) [iii] – Horst W. Doelle BIOTECHNOLOGY – Vol. I – Cell Thermodynamics and Energy Metabolism [iv] For thos with intrest in physics: W= w(Xi) is called th Work function and Xi is a tensor –notation indicating a n-deminsional space where X1 is denoting E, for energy produced by intracellular mitochondrias. W is defending on several factos such as functiron of alfa and beta receptors, cll membrane potential etc. T = t(Y1 ) is the tempraturefunction to be dealt with likewise.In a later paper we will deal with these functions that appears to be nonlinearl renasorfamtions. [v] Vanderpump Mark P: The epdidemiology of Thyroid disease. British Medical Bulletine, , June , 2011,http://bmb.oxfordjournals.org/content/99/1/39.full.pdf+html [vi] R., Heimdal, A., Karlsen, K., . . . Wyller, T. B. (2013). Ta faget tilbake! Tidsskrift for Den Norske Legeforening, 133(6), 655–659. Aarre, T. F. (2010). Manifest for Psykisk Helsevern. Oslo: Universitetsforlaget.

Written by Thyroid Patient Advocacy, August 16, 2004 Question: My doctor doesnt believe fibromyalgia has anything to do with hypothyroidism. She said that the rheumatologists she knows tell her that if fibromyalgia exists at all, the cause isn’t known. I’ve always felt that my fibromyalgiais related to low thyroid, but I don't have studies to show her. Are there any studies showing a connection that I can share with her? Dr. Lowe: Its obvious why your doctor doesn't understand the connection between hypothyroidism and what we call “fibromyalgia” symptoms, she gets her information from misguided rheumatologists. I trust that the rheumatologists she knows, as most, are well-intended. Largely, though, rheumatologists have been misled by the rheumatology researchers who originally and valiantly spearheaded the study of fibromyalgia. Those rheumatology researchers, throughout their thirty-year study of fibromyalgia, made a crucial mistake: they unquestioningly accepted as true a false belief perpetrated and perpetuated by the endocrinology specialty. That belief is that measuring the TSH and thyroid hormone levels infallibly identifies patients whose bodies are under-regulated by thyroid hormone. According to this false belief, if a patients levels are “normal,” then too little regulation by thyroid hormone can’t possibly be the cause of any symptom he or she complains of. The fact is, however, TSH and thyroid hormone levels are highly unreliable indicants of whose body is under-regulated by thyroid hormone. The levels are so unreliable that the testing should be abandoned as the standard method for identifying such patients. If your doctor is receptive, I suggest that she read two sets of information we recently posted to www.drlowe.com. In one, I refute the dogmatic belief of Dr. Richard Guttler that thyroid function testing is failsafe. In the other, I show that doctors are mistaken in their blind faith that thyroid function testing is reliable. (I provide more evidence in my forthcoming book Tyranny of the TSH. If you want the publisher to notify you when the book is available, send an email to McDPubCo@mcdowellpublishing.com with “Tyranny” in the subject line.) I also suggest that your doctor read my most recent brief summary of the evidence that too little thyroid hormone regulation is the main underlying cause of “fibromyalgia.” Please let her know, however, that we always follow that causative proposition with a qualification: typically, several metabolism-impairing factors contribute to patients fibromyalgia symptoms. The most common ones are nutritional deficiencies, unwholesome diet, low physical fitness, and drugs that slow metabolism. If your doctor reads my brief summary of the evidence, she may understand that the rheumatologists she knows are mistaken about fibromyalgia. She’ll also see that the UKs Peter Warmingham was correct two years ago when he succinctly wrote: “Fibromyalgia has been solved.”[1] Reference: [1] Warmingham, P.: Fibromyalgia has been solved. Fibro Focus Supporter, 3:1-3, 2002. November 13, 2002 Question: HELP! My 15-year-old has gone from being happy and healthy to being almost bed ridden. After our family doctor found a positive ANA blood test, we’ve gone to many doctors. This is what we know: She has Hashimotos thyroiditis and severe fibromyalgia. What we dont know is why her body is in so much pain all the time, to the point of her being in bed almost all day. At times she cries out in pain. Her body aches all over and at times she says it feels like a certain joint is being attacked. Her pain pills do no good. Her other symptoms are hair loss, not sleeping at night, swelling that comes and goes, and fatigue. Her exhaustion sometimes is so bad she can’t stand up and needs help walking to the bathroom. The severe fatigue we understand is from the Hashimotos thyroiditis. But since she tests negative for lupus and arthritis, why the awful pain? Dr. Lowe: I’m terribly sorry your daughter is suffering so severely. If the cause of her symptoms is hypothyroidism due to Hashimoto’s, her suffering is correctable and unnecessary. A subset of patients with thyroid hormone deficiency caused by Hashimoto’s has a lowered pain threshold. The susceptible patient perceives as painful stimuli that aren’t painful to other people. The pain results from too little thyroid hormone regulation of certain nerve cells. Some of the cells, mainly in her spinal cord, when under-regulated by thyroid hormone, release excess amounts of “substance P.” The excess substance P then amplifies the transmission of “pain” impulses in the central nervous system. Too little thyroid hormone regulation of other cells in the brain stem decreases the release in the spinal cord of a nerve transmitting substance called “noradrenaline.” The decreased noradrenaline in turn reduces the amount of opiates (morphine-like chemicals) released into the spinal cord. These opiates normally reduce the number of sensory impulses that enter the spinal cord and brain stem. When too few of the opiates are released, more sensory impulses make their way into the spinal cord and brain stem. As a result, the patient’s perception of pain is heightened. The combination of high substance P and low noradrenaline (and hence low opiates) causes the patient to perceive pain in the absence of painful stimuli. For example, the patient might perceive as painful the mere movement of some joints. She might experience pain from the pressure on her underside when she is sits or lies on a well-padded surface. And her pain threshold might be so low that she experiences aches and pains despite no apparent stimulus such as movement or pressure. My impression is that most doctors and researchers dont know that too little thyroid hormone regulation of cells in the brain stem and spinal cord can induce and sustain pain. When a hypothyroid patient is under-treated or denied treatment with thyroid hormone (the standard provisions of conventional medicine), and her main hypothyroid symptom is chronic, widespread pain, her doctor is likely to diagnose her pain as “fibromyalgia.” After the fibromyalgia diagnosis, conventional treatment will follow. This will entail various medications that dont correct the underlying cause of her pain (hypothyroidism) and that are largely ineffective. Through conventional care, her health is likely to deteriorate further over timepartly from her continuing hypothyroidism and partly from the adverse effects of conventional medications. To avert this from happening, I suggest that you and your daughter promptly abandon conventional medical care, and at the same time, get her under the care of an alternative doctor who'll competently treat her for her hypothyroidism. I wish her the very best. February 15, 1999 Question: Two years ago a car crashed into the back of my car and caused my neck to have a whiplash injury. I developed fibromyalgia within a month after the accident. I talked with my doctor about the accident maybe causing me to become hypothyroid. He told me that my fibromyalgia may seem like hypothyroidism, but they are two separate conditions. He doesn’t believe it is necessary to order tests for hypothyroidism. Instead, he insists that I continue my treatment with amitriptyline for fibromyalgia. He also says that a car wreck may cause fibromyalgia but not hypothyroidism. What bothers me is that my fibromyalgia symptoms are the same as my mother’s symptoms, but the same doctor gave her the diagnosis of hypothyroidism. Is it possible that my whiplash caused both fibromyalgia and hypothyroidism? Dr. Lowe: Keep in mind that many patients’ fibromyalgia symptoms are actually the symptoms of untreated or undertreated hypothyroidism. Your fibromyalgia may actually be hypothyroidism your doctor has failed to diagnose and properly treat. This is certainly possible because neck trauma often induces partial thyroid gland failure. The gland is then incapable of making and releasing enough thyroid hormones. As a result, the patient develops the symptoms of hypothyroidism. If the patient is a woman and her main symptoms are widespread pain and abnormal tenderness, she is likely to get the diagnosis of fibromyalgia. Tragically, her doctors are likely to ignore the hypothyroidism and have her to use various antidepressants – medications that are useless and dangerous for many fibromyalgia patients. Several research groups have found that trauma to the thyroid gland induces hemorrhaging in the gland.[1][2][3] The thyroid gland may be damaged even by adoctor examining it by touch (palpation). The palpation may cause inflammation and structural damage of the gland. The diagnosis of this condition is “palpatorythyroiditis.”[4] Directly relevant to your case is a study by Sehnert and Croft. They found that somepatients develop primary hypothyroidism after whiplash injuries.[5] They studied 101 consecutive whiplash patients. The basal temperatures of 86% of the patients were below normal. Of these patients, 30% had thyroid function test results indicating hypothyroidism. Of the 14% of patients whose temperatures were normal, 33% had abnormal laboratory thyroid test results. Sehnert and Croft diagnosed 30% of the101 patients as having post-traumatic hypothyroidism. They concluded that whiplash can result in a form of hypothyroidism due to direct injury to the thyroid gland. Another study done in Israel is relevant to your case. Buskila and colleagues found that a significant percentage of patients with neck injury develop fibromyalgia.[6] The researchers studied 102 patients with neck injuries and 59 patients with leg fractures. They examined all the patients for nonarticular (soft tissue) tenderness and the presence of fibromyalgia. None of the patients had a chronic pain syndrome before the trauma. Of the patients with neck injuries, 21.6% met the criteria for fibromyalgia. Only 1.7% of patients with leg fractures met the criteria. The incidence of fibromyalgia among patients with neck injuries was 13 times that of patients with leg fractures. Fibromyalgia developed at an average of 3.2 months after the trauma. Virtually all fibromyalgia symptoms were more common and severe among the 22 neck injury fibromyalgia patients. They had more tenderness, reported a lower quality of life, and had more impaired physical functioning than did patients without fibromyalgia. Because it is possible that your whiplash injury damaged your thyroid gland, you should have laboratory thyroid function tests. The results of the tests may provide evidence that you have hypothyroidism. If your present physician refuses to order the proper tests, I suggest you find another physician who will. It is critical that you do; undiagnosed and untreated hypothyroidism can have a devastating impact on one’s life. References: 1. Armstrong, W.B., Funk, G.F., and Rice, D.H.: Acute airway compromise secondary to traumatic thyroid hemorrhage. Archives of Otolaryngology: Head and Neck Surgery, 20(4):427-430, 1994. 2. Oertli, D. and Harder, F.: Complete traumatic transection of the thyroid gland. Surgery, 115(4):527-529, 1994. 3. Rupprecht, H., Rumenapf, G., Braig, H., and Flesch, R.: Acute bleeding caused by rupture of the thyroid gland following blunt neck trauma: case report. Journal of Trauma, 36(3):408-409, 1994. 4. Oertel, J.E. and LiVolsi, V.A.: Pathology of thyroid diseases. In Werner and Ingbar’s The Thyroid: A Fundamental and Clinical Text, 6th edition. Edited by L.E. Braverman and R.D. Utiger, New York, J.B. Lippincott Co., 1991, pp.609 610. 5. Sehnert, K.W. and Croft, A.C.: Basal metabolic temperature vs. laboratory assessment in “posttraumatic hypothyroidism.” Journal of Manipulative and Physiological Therapeutics, 19(1):6-12, 1996. 6. Buskila, D., Neumann, L., Vaisberg, G., Alkalay, D., and Wolfe, F.: Increased rates of fibromyalgia following cervical spine injury. A controlled study of 161 cases of traumatic injury. Arthritis and Rheumatism, 40(3):446-452, 1997. December 15, 1998 Question: I have both fibromyalgia and hypothyroidism. Recently, an endocrinologist answered a question I sent to him through the Internet. In his answer, he made the following statement: “Muscle pain is a very unusual MAJOR symptom of hypothyroidism…” Will you comment on his statement? Dr. Lowe: This is not the first time I have heard this statement made by an endocrinologist. The statement is false. This misbelief of theirs has resulted from their restricting hypothyroid patients to “replacement” dosages of thyroid hormone dosages that keep the TSH level within the range of “normal.” Many hypothyroid patients have widespread and severe muscle pain. And many of these patients continue to have muscle pain as a residual symptom even after their endocrinologists place them on (and restrict them to) “replacement” dosages of thyroid hormone. When a patient continues to complain of muscle pain, the endocrinologist typically concludes that the pain must be caused by “something other than a thyroid hormone deficiency.” The endocrinologist thinks that if the pain were caused by a thyroid hormone deficiency, then it would disappear since the patient is on a “replacement” dosage. The replacement dosage the endocrinologist refers to is a dosage that “replaces” the TSH level back to normal. Unfortunately, in general, this dosage does not adequate accomplish what’s really important==replace the patient’s tissue metabolism back to normal. Typically, the endocrinologist refers the patient who continues to complain of muscle pain to a rheumatologist for a fibromyalgia evaluation. We have had many years of experience treating hypothyroid patients who also meet the criteria for fibromyalgia. The distinguishing symptom of fibromyalgia, of course, is muscle pain. When we permit these patients to use TSH-suppressive (but non-thyrotoxic) dosages of thyroid hormone, virtually all of them have complete relief from their muscle pain. These patients’ complete relief from muscle pain is not merely an anecdotal observation; we have documented the finding by objective measures under both clinical and experimental conditions. For some patients, it is necessary that we also use effective physical treatment (especially trigger point myofascial therapy) before the pain completely disappears. It is important to note, however, that without the TSH-suppressive dosages of thyroid hormone, the patients’ pain persists despite the physical treatment. By forcing patients to use only what they call “replacement” dosages of thyroid hormone, endocrinologists deprive themselves of valuable clinical experience. In effect, they rob themselves of the opportunity to witness the relief of hypothyroid patients’ muscle pain through sufficiently high dosages of thyroid hormone. As a pain management specialist, fibromyalgia researcher, and thyroidologist, I offer firm testimony: The belief of endocrinologists that muscle pain is not a common major symptom of hypothyroidism is simply a self-imposed and self-perpetuated false belief. December 13, 1998 Question: I have both hypothyroidism and severe fibromyalgia. My fibromyalgiasymptoms improved when my family doctor let me increase my Synthroid (T4) dose from 0.10 mg to 0.15 mg per day. However, he won’t let me increase the dose any further. I also see a nutritional doctor. This doctor recently tested my blood for betacarotene and found that it is high. I take only 25 mg of beta carotene each day. Despite the low intake of beta carotene, the nutritional doctor suggested that I reduce my dose even further to get my blood level down to normal. I haven’t lowered the dose yet. My reason is that I read somewhere that hypothyroid patients have high beta carotene levels in the blood. Does my high blood level of betacarotene mean that I am not taking enough Synthroid? Or should I lower my betacarotene dose? Dr. Lowe: First, your fibromyalgia symptoms are most likely nothing more than hypothyroid symptoms. This is indicated by the improvement in your fibromyalgia symptoms with the increased dosage of thyroid hormone. We find that fibromyalgia symptoms completely disappear when most hypothyroid patients take dosages of thyroid hormone that suppress the TSH level. Dosages that suppress the TSH level are larger than the “replacement” dosages mandated by conventional endocrinologists. In most cases such as yours, the elevated blood beta carotene level results from the hypothyroid patient taking too little thyroid hormone. High serum levels of beta carotene are found in many hypothyroid patients. In fact, a high carotene level is the best-known example of abnormal vitamin metabolism in hypothyroidism. There are two mechanisms that account for the high carotene levels. One of these is not a serious concern, but the other raises ominous possibilities. The first mechanism is reduced conversion of carotene to vitamin A in hypothyroidism. The reduced conversion leaves more carotene to circulate in the blood. This may prove annoying in that it may cause a slightly yellowish tint of your skin, especially of your palms and soles. The second mechanism is elevated “lipoproteins.” Lipoproteins are compounds in the blood that contain fat and protein. The low density lipoproteins (also called LDL) are rich in cholesterol and triglycerides. The compounds are called “bad” lipoproteins for good reason: When these lipoproteins elevated (as they are in untreated and under-treated hypothyroid patients), they are a risk for cardiovascular disease. Carotene is transported in the blood by lipoproteins, and a high carotene level indicates that the patient also has high lipoprotein levels. There is considerable evidence that the dosage of thyroid hormone that conventional doctors allow hypothyroid patients to use is too low to reduce lipoprotein levels to normal. As a result, patients taking these inadequate thyroid hormone dosages may also have elevated circulating carotene levels. In that the inadequate dosages increase the patients’ risk for cardiovascular disease, heart attacks, and strokes, the conventional treatment protocol is a public health menace. Patients and informed physicians should aggressively oppose the conventional protocol being imposed on hypothyroid patients. (For more detailed information on this, see my rebuttal to thyroidologist Dr. Robert Volpe.) In making treatment decisions, your physicians and you must consider the peculiarities of your case. In doing so, I would urge them and you to consider a potentially serious possibility==that your elevated beta carotene level indicates that your thyroid hormone dosage is too low to adequately protect your cardiovascular health. A higher dosage may provide cardiovascular protection, and it may also lower your blood carotene level. November 5, 1997 Question: My wife had cancer (papillary w/ follicular variants) of the thyroid. Her thyroid was removed in March of 1997, and so she’s now on Synthroid, 0.150 mg daily. She also underwent post surgical oblation with I-131, I think it was 120 millicuries (something like 80% of the max dose which if I recall was 150 mc). Anyway, she has had CFIDS/CFS symptoms since 1991, and pretty severe FM. Can she be put on your protocol and if so, what adjustments would have to be made given the missing gland? Her cancer prognosis is great, no sign of metastases. Dr. Lowe: If your wife meets the criteria for fibromyalgia, the medical history you’ve given indicates that she is a good candidate for our treatment protocol. You may know that surgical removal and radioactive iodine (I-131) treatment of the thyroid gland are “antithyroid” therapies. This means that they decrease the amount of thyroid gland tissue. A consequence is that the amount of thyroid hormone produced in the thyroid gland, leaving the patient hypothyroid to some degree. Anyone who has undergone antithyroid therapy should take thyroid hormone orally, as your wife does. The thyroid hormone medication serves two purposes. First, it decreases the likelihood of recurrence of the cancer. Second, it should compensate for the thyroid hormone the patient is no longer able to produce internally. Unfortunately, many of such patients take too little thyroid hormone to maintain normal metabolism in the tissues that give rise to fibromyalgia symptoms. For many people, fibromyalgia begins after the antithyroid treatment, despite the use of thyroid hormone medication at the typically prescribed dosage. These patients usually join the fibromyalgia roles as victims of the “tyranny of the TSH.” By this, I mean that their doctors dictate the patients’ thyroid hormone dosages according to the patients’ serum TSH levels. The TSH level tells us only how the anterior pituitary gland is responding to the dosage of thyroid hormone. It tells us absolutely nothing about how all the other tissues of the body are responding. Yet, there is considerable variability in how different tissues respond. (A recent paper that deals with this phenomenon is: Hector, F., et al.: “Replacement therapy for hypothyroidism with thyroxine alone does not ensure euthyroidism in all tissues, as studied in thyroidectomized rats. Journal of Clinical Investigation, vol. 96, pages 2828-2838, 1995.) As a result, in many patients, certain metabolically understimulated tissues give rise to fibromyalgia symptoms. Many years of clinical and experimental experience with hypothyroid fibromyalgia patients have made one thing clear: few improve or recover from their fibromyalgia symptoms with a Synthroid (or other T4 product) dosage as low as 0.15 mg. We have had a high rate of improvement and recovery with hypothyroid fibromyalgia patients. Our protocol is to (1) ignore the patient’s TSH level, (2) increase her T4 dosage based on the metabolic responses of tissues other than the anterior pituitary (which secretes TSH), and (3) have her take nutritional supplements and exercise to tolerance. We’ve carefully monitored patients for the adverse effects many doctors fear from patients taking relatively high dosages (0.2 mg-to-0.4 mg) of thyroid hormone. The results do not justify the fear. That your wife had fibromyalgia symptoms some six years prior to having antithyroid treatment may indicate that she had a thyroid hormone deficiency before developing thyroid cancer. If she had thyroiditis, which increases the risk of thyroid cancer, she could have had fibromyalgia symptoms from an associated thyroid hormone deficiency. On the other hand, she may have had impaired responsiveness of fibromyalgia-related tissues to normal thyroid hormone levels. If this is this case, she will most likely require T3 rather than T4 (and a fairly large dosage). Thyroid Patient Advocacy

An Interview with David Derry, M.D., Ph.D. The History of Thyroid Testing, Why the TSH Test Needs to Be Abandoned, and the Return to Symptoms-Based Thyroid Diagnosis and Treatment Written by Mary Shomon / Thyroid Patient Advocacy Almost every conventional discussion of thyroid disease focuses on the use of the Thyroid Stimulating Hormone (TSH) as the diagnostic “gold standard” for thyroid disease. The TSH is used almost exclusively by most conventional physicians as the means of diagnosing thyroid disease, irrespective of symptoms. Typically, if the TSH level is above the normal range, a patient is diagnosed as hypothyroid, and TSH levels below normal range are interpreted as hyperthyroidism. But is the TSH test and the reference “normal range” accurate? Should thyroid disease diagnosis be based primarily on this one test? Some experts say no. Dr. A P Weetman, professor of medicine, wrote in the article “Fortnightly review: Hypothyroidism: screening and subclinical disease ,” which appeared in the 19 April 1997 issue of the British Medical Journal, the following groundbreaking statement: “...even within the reference range of around 0.5-4.5 mU/l, a high thyroid stimulating hormone concentration (>2 mU/l) was associated with an increased risk of future hypothyroidism. The simplest explanation is that thyroid disease is so common that many people predisposed to thyroid failure are included in a laboratory’s reference population, which raises the question whether thyroxine replacement is adequate in patients with thyroid stimulating hormone levels above 2 mU/l.” In response to Dr. Weetman, David Derry M.D., Ph.D., a thyroid expert and researcher, based in Victoria, British Columbia, responded, saying: “Why are we following a test which has no correlation with clinical presentation? The thyroidologists by consensus have decided that this test is the most useful for following treatment when in fact it is unrelated to how the patient feels. The consequences of this have been horrendous. Six years after their consensus decision Chronic fatigue and Fibromyalgia appeared. These are both hypothyroid conditions. But because their TSH was normal they have not been treated. The TSH needs to be scrapped and medical students taught again how to clinically recognize low thyroid conditions.” This provocative response was how Dr. Derry came to the attention of many thyroid patients, and interviewer Mary Shomon, About’s thyroid guide. In this interview, Dr. Derry shares his fascinating and innovative ideas about why he believes the TSH test needs to be abandoned. This interview was conducted in July of 2000. Mary Shomon: First, Dr. Derry, can you tell us a little bit about your medical background, and interest in thyroid testing and treatment? David Derry: I have always been interested in Medical research. I graduated with a Medical Degree from the University of British Columbia, Canada in Vancouver in 1962. I interned at the Toronto General Hospital. From there I went to McGill University and went into a four year program to get my PhD in biochemistry and more specifically in Neurochemistry at the institute set up by Wilder Penfield called the Montreal Neurological Institute in Montreal. In 1967 I graduated with a PhD in biochemistry from McGill. I was hired by the department of Pharmacology at the University of Toronto Medical School as an assistant professor. For five years I did basic biochemical research and taught medical students, dentistry students and pharmacy students. Not long after I arrived in Toronto I was became a Scholar of the Medical Research Council of Canada. That is to say, my salary was paid by The Medical Research Council of Canada to do pure research for five years. At the same time I worked week-ends in charge and the only physician in a large 900 bed psychiatric hospital called the Lakeshore Psychiatric Hospital. Meanwhile about then (1970) I had a rearrangement of my domestic status. I ended up marrying my present wife and by this gained three more children. I had two of my own. All children were between 4 and 9 years old. There was no way that the salary of an assistant professor in Pharmacology at the University of Toronto was going to be able to pay to raise five small children. After the legal aspects had been settled my wife and I, the five children and a large Labrador retriever boarded a 747 for Victoria British Columbia. Within two weeks I started a general practice. When I came back into General Practice I had in mind a saying I attribute to Dr. Wilder Penfield which was (paraphrasing) “If you listen to a patient carefully the patient will tell you the diagnosis and if you listen even more carefully they will tell you the most appropriate treatment”. Before I went back into practice I had taken courses in interpersonal relationships and how to communicate and listen better. Since I entered General Practice I have taken more courses in personal development. My idea was to learn more and more how to listen carefully and how to get my personality (ego) out of the way of the conversatio with the patient. Because I was armed with this approach I developed, I have been able to learn much in the last 28 years in practice. After about 3-4 years in practice I thought I would start to do my own research. I started with Vitamins. Amongst many other topics, I taught Vitamins at the University of Toronto and when Dr. Linus Pauling’s book on Vitamin C and Cancer came out in 1970 I was asked by the Faculty of Medicine to present the essential material of the book to about 300 faculty members and students. Therefore, vitamins, their prophylactic and therapeutic use was a good place for me to start to investigate. So I investigated the use of vitamins for all manner of disease. Eventually after about 10 years I had fairly well exhausted every aspect of the therapeutic use of vitamins I could think of. By then I knew what you could do and couldn’t do with vitamins. Most of the patients were only too glad to help me with this and the ones who got better were very grateful. Since then I have slowly over the last 15-20 years developed an interest in thyroid problems. There are reasons for my interest in thyroid that are too long to tell. Gradually I obtained copies of all the relevant thyroid literature back to the 1883 Committee on Myxedema. I have a huge library on the thyroid literature consisting of about 5000 reprints and books. All of the old textbooks I copied and have them in my library for my use. All of this is computerized of course. There are other aspects of my medical and biological training in my CV. ( See Dr. Derry’s Biographical Information and Chronological Curriculum Vitae ) The consensus of thyroidologists decided in 1973 that the TSH was the blood test they had been looking for all through the years. This was about two years after I started practice. Having been taught how to diagnose hypothyroid conditions clinically I was in a position to watch to see what the relation of the TSH was to the onset of hypothyroidism. What I found was many people would develop classic signs and symptoms of hypothyroidism but the TSH was ever so slow to become abnormal, rise and confirm the clinical diagnosis. Sometimes it never did. Finally I began treat patients with thyroid in the normal manner I was taught. I could not see why I had to wait for the TSH to rise for me to be able to treat them. The main ingredient of thyroid hormone, which distinguishes it from other molecules of similar size (molecular size), was the element which made thyroid hormone namely iodine. So I did a thorough search of the literature on iodine. This review led me to try to use iodine and thyroid therapeutically. The TSH had caused all research on the therapeutic use of both of these substances to stop dead. My biochemical and pharmacological background has allowed me to search in areas of the literature that are impossible for a normal physician or even a specialist to explore. If you remember it was a long time before the medical profession admitted that there were two new diseases to appear in the world that were not there before. Chronic fatigue and fibromyalgia were non-existent before 1980. This is seven years after the 1973 consensus meeting. So where did these two new diseases come from? The symptoms and signs of chronic fatigue and fibromyalgia were described in the literature in the 1930’s as one way that low thyroid could be expressed. Treated early it was easily fixed with thyroid in adequate doses. But even then the clinicians had noticed that if a patient has low thyroid (chronic fatigue and fibromyalgia) for too long then it became more difficult to reverse all signs and symptoms regardless of what they were. Mary Shomon: Why do you think that thyroidologists have decided that the TSH test is the most useful — or in many cases – the only test for thyroid problems, versus a patient’s clinical symptoms? How do you think this has come to be considered the “gold standard” for thyroid diagnosis and management? David Derry: The thyroidologists have been looking for a reliable test for thyroid function since the beginning of the century. The first important ones were the Basal Metabolic Rate, the cholesterol and the creatine phosphokinase. (CK). These were used mainly up to about 1960. If you had a high cholesterol in the first half of the century you got thyroid to lower it to normal. Details of using this method of treatment were still described in the 1950’s. The Basal Metabolic rate became the fad in the 30s and 40s and almost every office had a machine to measure it. It was quite good but subject to difficulties of interpretation and interference by emotional factors. However it still remains the only test that actually measures the effects of thyroid medication on the human body. In the 1940’s radioactive iodine became available from the Tennessee Valley Atomic Energy Complex. Hence the metabolism of iodine could be studied more closely. The radioactive iodine uptake by the thyroid became a frequently used test, which was said to be infallible like all the others when they first arrived. Every time there was a new test it was declared to be reliable for telling if a person was hyperthyroid or hypothyroid, but as with every previous test it turned out to not be clinically applicable in all cases. In the 1960’s when I was studying medicine the PBI (Protein bound iodine) was heralded as the only test necessary, when it was low you had hypothyroidism and when it was high you had hyperthyroidism. This was written in some of the textbooks of the time. Eventually this test went the way of the rest — useful sometimes– but doesn’t always agree with the clinical findings. Next came the T4 or total thyroxine in the blood that is the free and the protein bound thyroxine measured together. This was also hailed as far superior to the PBI, but it too went the way of the rest of the tests –as not being reliable enough. Finally the TSH arrived in the late 60s and was boasted about as the final answer. The TSH was not only able to deliver all the thyroid diagnoses but it could be used as well for monitoring therapy. Over the following twenty years the TSH was made more and more sensitive and because of these improvements it was even more thought that it was the total answer for thyroid diagnosis and treatment. However as the TSH was so sensitive to orally-given thyroid hormone it meant literally everyone was going to end up with a low dose by comparison with previous doses. The new doses were about a third of the dose that had been found to be clinically effective for every patient for eighty years prior to the TSH. The TSH had a ring of scientific rigor for those who have a smattering of knowledge about thyroid metabolism. It was part of the pituitary feed back mechanism for monitoring the output of the thyroid gland. There is no doubt that it does accomplish this job. But unfortunately the TSH value has no clinical correlation except at absolute extremes with the clinical signs or symptoms of the patient. The reasons for this are complex and I only want to discuss one aspect but there are other important factors. To start with the thyroid metabolism is controlled locally in the tissue by each organ. That is the brain has one mechanism for controlling the amount of thyroid available to the brain but it is different from other tissues such as the liver. There are many mechanisms by which each tissue controls the amount of thyroid hormone which gets into the tissues. But to discuss one: there is an enzyme in the tissue which deiodinates (takes one iodine off the thyroxine T4) and makes T3 or triiodothyronine. These enzymes are called deiodinases. Every tissue has different types of deiodinases. To just give you one example: If you starve animals and study the deiodinases in the brain and liver you find that the activity in of the brain deiodinases go up by 10 times while at the same time the liver deiodinases go down–not up. This mechanism is obviously meant to preserve the functioning of the brain under starvation conditions and not metabolize too much thyroid hormone in the liver. Therefore the control of thyroid metabolism is in every individual tissue. The problem with this is– if a tissue needs more (such as the brain with depression) there is no way for the brain to signal the thyroid that it needs more sent up to it. The thyroid merrily goes on putting out the same amount of thyroid hormone. So the patient can have symptoms related to low thyroid in the brain (for example) but the thyroid doesn’t do anything about it. But if you give thyroid hormones in an adequate dose the brain symptoms will disappear. Meanwhile the other tissues and organs adapt to the increased circulating hormones that you have used to fix the brain with. The adaptation of the tissues to different levels of circulating hormones has been shown in the literature. The symptoms of low thyroid, which are numerous and variably expressed, can be related to any organ or system in the body and partly depends on the person’s genes. But because of the all inclusiveness of the TSH medical students are not taught or only superficially taught the symptoms of low thyroid. The TSH was “scientific” and held all the answers to thyroid disease. If you have not lived through several versions of the ultimate test for thyroid then it is harder to grasp this phenomenon. Mary Shomon: If, as Dr. Weetman suggests, the laboratory’s reference range for “normal” TSH includes people who are in the process of developing hypothyroidism, do you feel that the reference range itself should be recalculated? David Derry: This suggestion agrees with what I have been saying namely that the TSH can lag a long way behind the appearance of low thyroid symptoms. One clear case I remember is a lady who started to lose her hair at age 26 and had lost it all by the time she was 35 but the TSH did not turn up until she was 48. Then her TSH rose very high for the first time. (The TSH was several hundred). She was being treated for a heart condition at the time but when the cardiologist saw the TSH, he said just treat the low thyroid condition and her heart problems will go away. He was right. Her hair has not grown back yet. She has been taking thyroid for about 1 year now. So her TSH lagged her symptoms and signs by 22 years. In some cases the TSH appears never to turn up and confirm the clinical diagnosis. It is difficult to visualize using the TSH when, as Dr. Weetman has said, people can be low thyroid with a normal TSH. The truth is that there is no relationship between the TSH and how people feel. Dr. Anthony Toft has stated this in the bible of thyroidology Werner and Ingbar’s “TheThyroid” in 1991. I have quoted this reference in my response. Dr Weetman has also said in a response on line to the BMJ article of May 2000 article by Denis StJ. O’Reilly on “Thyroid function tests_time for a reassessment” that he thinks that a patient can have a profound hypothyroidism without any signs or symptoms. This is incorrect. To follow the TSH while you are treating someone for low thyroid is also going to lead to under-treating the patient. The pituitary cells, which have TSH in them, are the most sensitive cells in the body circulating thyroid hormone. Therefore when one treats hypothyroidism by following the TSH and trying to make it normal the pituitary cells are happy but the rest of the body is short-changed by a considerable amount I also mentioned in my response to the same article that the normal dose arrived at by all the best clinicians in the world over 80 years of experience was between 200 and 400 micrograms of Eltroxine. But some need more. Currently treated patients average about 100 micrograms which is about a third of the dose that has been known for a century to help patients return to normal. Long term studies found no difference in any disease between normal people and people taking the thyroid at the higher doses. Recent studies have confirmed this opinion. The tail end of this approach of the method of treating hypothyroidism was documented by Professor R. Hoffenberg in the introduction to his two part article on hyperthyroidism in the British Medical Journal 1974. Reflecting on a life-time of treating thyroid patients, he mentions that his personal record of amount of thyroid he needed to give to a patient to make them feel right was 29 grains of desiccated thyroid. which represents approximately 1700 mgs of desiccated thyroid which would be equal to about 2900 micrograms of Eltroxine. This highly-respected thyroidologist had spent a lifetime treating thyroid patients must have prepared the paper and had it accepted during 1973 when the consensus meeting on the TSH occurred. This all means even if the chronic fatigue patient does have an abnormal TSH the treatment will be inadequate to make them well again. The clinicians of the past (before the TSH) were astute and very observant and were able to diagnose and treat hypothyroidism correctly without the TSH for 80 years– why do we need it now? They would be aghast at the total missing of the diagnosis of chronic fatigue and fibromyalgia. The treatment of thyroid cancer before the TSH involved giving the maximally tolerated dose of thyroid in order to stop the cancer. It was termed then a sub-toxic dose. That is the dose of thyroid would be raised until some toxic symptoms occurred then the dose would be lowered slightly to remove the symptoms such as sweating too much or tachycardia. The present treatment of thyroid cancer patients only get enough to suppress the TSH which is usually a much lower amount. Nothing untoward happened to these patients of the era before the TSH because of this higher thyroid dose. Mary Shomon: You indicated that you feel chronic fatigue and fibromyalgia are both hypothyroid conditions. There are some physicians who feel that these two conditions are manifestation of difficult to diagnose hypothyroidism, and yet other studies claim there is no relationship. Can you explain why you feel there is a connection among these conditions? David Derry: For many years the literature (before the TSH) supported the fact that if your symptoms responded to thyroid hormone you were low thyroid but especially if when you took the person off the thyroid and their symptoms returned. My own patients who develop chronic fatigue or fibromyalgia I treat them with thyroid and all –and I mean all– of their symptoms disappear. If I stop the thyroid or if they stop it for some reason all the symptoms start to slowly come back over the following months. You might ask do I do thyroid function tests? The answer is yes if for no other reason that I am curious to know what they look like in the face of the patient’s obvious clinical diagnosis. The other patients who come to me from outside my practice respond roughly in proportion to how long they have had it. But I have had lots of pleasant surprises of people badly disabled by fibromyalgia or chronic fatigue for six years or more who slowly over 6 months to a year their symptoms completely disappear. It is of course a delight to see this happen. Mary Shomon: Do you feel that TSH – or TRH, T4/T3, antibodies, or Reverse T3 – tests have any place in thyroid diagnosis and management, or do you believe that diagnosis and treatment should be solely based on symptoms. David Derry: One place that TSH has been useful is testing new-born babies. The TSH which is backed up by the free T4 or the Total T4 tells you quickly that the baby has a serious problem which must be treated immediately. This does not say that the TSH is infallible in this instance but at least when it is abnormal then you know you have a serious problem. A phenomenal number of thyroid function tests are ordered by physicians today but it is unlikely that it has helped that much as the physicians have been ignoring the symptoms of low thyroid which so many patients complain about. Mary Shomon: What are the most common clinical hypothyroidism symptoms you’ve found most useful in making a diagnosis? David Derry: I gave a lecture on thyroid about three years ago and discussed this topic. The signs and symptoms list continues to grow as I learn more. Most people will have some form of fatigue but there is a group that are high output (read will-power) and low thyroid. These patients are not always thin. A good example I had was a 19-year old ballet dancer, while dancing for a national Ballet company, started to see a counselor for depression. She continued to dance with the company. There is no way a person that physically healthy and that age with no previous history should be depressed except by low thyroid. But suspicions would be heightened when you know her mother and her sister have low thyroid conditions. Her TSH was raised above normal but the cure was the same regardless of whether the TSH was abnormal or not. She has been completely well since her low thyroid was corrected. All ballet dancers have enormous drive and self-control therefore they can ignore many symptoms and carry on. So people with strong will power can ignore many symptoms and signs for a long time. To quote Dr. George Crile on hypothyroidism from his textbook in 1932 on The thyroid and its diseases. “In the advanced stage of the disease the patient may complain of almost any symptom which can result from a low metabolism. A summary of the literature discloses that symptoms referable to every organ in the body have been attributed to thyroid deficiency and have been relieved by the administration of thyroid extract.” I now have a huge organized list of hypothyroid symptoms which I will not burden you with. Initially it was made for my lecture but I have been adding to it slowly since as I witness new signs and symptoms disappear with therapy. People who have had terrible childhood experiences (sexual abuse, physical abuse, personal tragedies etc) for whatever reason have altered thyroid metabolism. They are more complex to treat. They are different from everyone else biochemically and pharmacologically. The blame for most of their residual difficulties is not with their brains and minds but with their chemistry. I believe also other areas of their biochemistry are not normal. I don’t think this has been generally recognized yet. Mary Shomon: What type of thyroid hormone replacement therapy do you favor? Levothyroxine, levothyroxine plus T3, or natural thyroid hormone replacement, and why? David Derry: I use any of the above. In Canada we have only Eltroxine (levothyroxine) or desiccated thyroid (Parke-Davis). T3 is available through specialty pharmacies but is not as readily available as in the US. If I don’t get the response that I am looking for, I will often switch either way in order to try and make the patient better. Mary Shomon: Are you practicing currently? How can patients arrange to see you? Do you do phone consults? David Derry: I have no intentions of retiring in the near future. Since I gave my two lectures three years ago on Breast cancer and Thyroid I have had close to 2000 new patients. Fortunately for me I have been able to help most of them so I don’t have to go on seeing them. A fair number of patients come quite far. I have a bunch of patients who come from Alberta and one of these is a young lady from Calgary who flies to Victoria for each of her appointments. All who want to come can do so by booking through the office, at 250 478-8388. I would be too flooded to answer much over the phone. Also I am sure I couldn’t diagnose and or treat anyone without meeting with them I need to follow people for several months after seeing them– but not often after that–as the thyroid works so slowly that you have to give it time. At present I am writing a book on breast cancer. Hopefully it will be finished by year-end or before. After the breast cancer book I will write one on this topic. Contact Information: David M. Derry M.D., Ph.D. E-Mail dderry@shaw.ca See Dr. Derry’s Biographical Information and Chronological Curriculum Vitae