Utility of systematic TSHR gene testing in adults with hyperthyroidism lacking overt autoimmunity and diffuse uptake on thyroid scintigraphy

Summary Objective Patients with hyperthyroidism lacking autoimmune features but showing diffuse uptake on thyroid scintigram can have either Graves’ disease or germline activating TSH receptor (TSHR) mutation. It is important to identify patients with activating TSHR mutation due to treatment implication, but the overlapping clinical features with Graves’ disease make it difficult to discriminate these two conditions without genetic testing. Our study aimed to assess the potential of systematic TSHR mutation screening in adults with hyperthyroidism, showing diffuse uptake on thyroid scintigraphy but absence of TSH receptor antibodies (TRAb) and clinical signs of autoimmunity. Design A cross‐sectional study of Caucasian adults with hyperthyroidism, managed at three endocrine centres in the South West, UK, from January 2006 to April 2017. Methods We recruited 78 adult Caucasian patients with hyperthyroidism showing diffuse uptake on 99mTc‐pertechnetate thyroid scintigraphy but without TRAb and other autoimmune clinical features of Graves’ disease (such as thyroid‐associated ophthalmopathy or dermopathy). Genomic DNA of these patients was analysed for variants in the TSHR gene. Results Genetic analysis identified 11 patients with four variants in TSHR [p.(Glu34Lys), p.(Asp36His), p.(Pro52Thr) and p.(Ile334Thr)]. None of these variants were pathogenic according to the American College of Medical Genetics and Genomics guideline. Conclusions Activating TSHR mutations are a rare cause of nonautoimmune adult hyperthyroidism. Our study does not support the routine genetic testing in adult patients with hyperthyroidism showing diffuse uptake on scintigraphy but negative TRAb and lacking extrathyroidal manifestations of Graves’ disease.


Funding information
This study was funded by Royal Devon and Exeter Hospital Small Grant Scheme and Society for Endocrinology Early Career Grant awarded to KAP. KAP has a postdoctoral fellowship funded by the Wellcome Trust (Grant Number 110082/Z/15/Z).

Summary
Objective: Patients with hyperthyroidism lacking autoimmune features but showing diffuse uptake on thyroid scintigram can have either Graves' disease or germline activating TSH receptor (TSHR) mutation. It is important to identify patients with activating TSHR mutation due to treatment implication, but the overlapping clinical features with Graves' disease make it difficult to discriminate these two conditions without genetic testing. Our study aimed to assess the potential of systematic TSHR mutation screening in adults with hyperthyroidism, showing diffuse uptake on thyroid scintigraphy but absence of TSH receptor antibodies (TRAb) and clinical signs of autoimmunity.

Conclusions:
Activating TSHR mutations are a rare cause of nonautoimmune adult hyperthyroidism. Our study does not support the routine genetic testing in adult patients with hyperthyroidism showing diffuse uptake on scintigraphy but negative TRAb and lacking extrathyroidal manifestations of Graves' disease.

K E Y W O R D S
Graves' disease, hyperthyroidism, thyroid scintigraphy, TRAb, TSHR

| INTRODUC TI ON
Activating TSH receptor (TSHR) mutations are a rare cause of nonautoimmune hyperthyroidism, although the true prevalence in adults is unknown. [1][2][3] The majority of these cases are familial, with autosomal dominant patterns of inheritance, but mutation can also arise de novo. 1,2,4 Identification of these patients has important clinical implications. Hyperthyroidism due to activating TSHR mutations does not remit following antithyroid drug treatment, and patients usually require total thyroidectomy or radioiodine ablation as a definitive treatment. 1,2 A genetic diagnosis at the time of first presentation could, potentially, prevent unnecessary and prolonged treatment with antithyroid drugs, as well as reducing the associated morbidity and cost. This would also enable screening immediate family members, leading to earlier identification and treatment of undiagnosed hyperthyroidism.
It is difficult to identify patients with hyperthyroidism due to activatingTSHR mutation, as their clinical features show significant overlap with Graves' disease. Similar to Graves' disease, presentation is highly variable, from subclinical hyperthyroidism to severe hyperthyroidism, 1,2,5 with ages at presentation ranging from the neonatal period up to 60 years, 1,6 and has a diffuse uptake on thyroid scintigraphy. 2 A family history of thyrotoxicosis is not always present in patients with activating TSHR mutation due to variable penetrance and seen in 30% of patients with Graves' disease 7 ; thus, it is not very useful in discriminating these two aetiologies. Contrary to Graves' disease, patients with an activating TSHR mutation do not have TSHR antibodies (TRAb) or extrathyroidal autoimmune clinical manifestations such as thyroid-associated ophthalmopathy and dermopathy. 1,2 However, around 3%-5% patients with Graves' disease also do not have TRAb when analysed with the second-or third-generation assays, 8,9 and only about 25% will have extrathyroidal manifestations, such as thyroid-associated ophthalmopathy. 10 Altogether these data suggest that it is difficult to identify patients with activating TSHR mutation in routine clinical practice without systematic genetic testing. However, utility of this approach is not known. Therefore, we aim to assess the utility of systematic genetic testing for TSHR mutations in adults with hyperthyroidism showing diffuse uptake on thyroid scintigraphy but TRAb negative and lacking extrathyroidal manifestations of Graves' disease.

| Study participants
We recruited patients from three endocrine centres (Exeter, Plymouth and Torbay) in the South West, England (UK). The inclusion criteria were as follows: age at recruitment >18 years, biochemically confirmed overt or subclinical hyperthyroidism according to the American Thyroid Association guideline, 11 absence of TRAb, diffuse uptake on the thyroid scintigraphy and Caucasian ethnicity. We excluded patients with any extrathyroidal manifestations of Graves' disease, such as thyroid-associated ophthalmopathy or dermopathy, and patients with any other known cause of hyperthyroidism (eg, toxic nodular disease, thyroiditis and drug-induced thyrotoxicosis).

| Study recruitment
We identified 275 patients from the clinical records who had thyroid scintigraphy and had absence of TRAb from all three centres over 11 years (2006-2017). Local endocrinologists performed TRAb and thyroid scintigraphy at presentation as part of the routine clinical care. The research team reviewed all thyroid scintigraphy to identify patients with diffuse uptake. The clinical records were reviewed to assess the eligibility of these patients. Of the initial 275 potential participants identified, three had died, 41 had decreased or no uptake on scintigraphy, 98 had patchy/localized uptake, two had Graves' ophthalmopathy, and four had drug-related thyrotoxicosis, and they were therefore excluded. All 127 patients who fulfilled the entry criteria were approached and invited to take part in the study, and 78/127 (61%) were recruited (49 refused or were unable to be contacted directly). We estimated the frequency of activating TSHR mutation in adults with hyperthyroidism lacking overt autoimmunity and diffuse uptake on thyroid scintigraphy to be ~4.5% based on two previous studies. 12,13 This provided a sample size of 67 to find at least one individual with a mutation at P < 0.05.

| DNA and data collection
We collected one-off blood or saliva sample for DNA extraction.

| Data analysis
Statistical analysis was conducted using StataSE ®

| Study cohort characteristics
The clinical characteristics of the study cohort are shown in Table 1.
The median age at diagnosis of hyperthyroidism was 52 years, 76% were female, and 42% had a palpable diffuse goitre. None had thyroid-associated ophthalmopathy, dermopathy or TRAb, all had diffuse uptake on thyroid scintigraphy, and 35% had a family history of thyrotoxicosis.

| Analysis of TSHR variants
Genetic analysis identified 11 patients with four variants inTSHR ( Table 2). None of these variants were novel or pathogenic accordingly to the ACMG guideline. 14 p.(Glu34Lys), p.(Asp36His) and p.(Pro52Thr) were definitely benign according to the ACMG guideline. All the variants were outside the mutational hot spot for the disease (transmembrane domain) and present in the larger control population (gnomAD version 2.0, n ~ 137 000) 15 at a frequency incompatible with rare monogenic disease (Table 2). Furthermore, two of these variants, p.(Asp36His) and p.(Pro52Thr), were seen previously in patients with Graves' disease with TRAb and ophthalmopathy. [16][17][18] The p.(Glu34Lys) variant was seen in an individual with congenital hypothyroidism and the probands' mother with Graves' disease ( Table 2). 19 Both p.(Glu34Lys) and p.(Asp36His) had in vitro evidence of normal or low activity. 18,19 These data together suggest that these are not activating pathogenic variants. TA B L E 1 Clinical characteristics of the study cohort spot, not conserved and seen in five patients in gnomAD (Table 2).

| Analysis of TSHR variant p.(Ile334Thr)
Based on the frequency of this variant in gnomAD, for this variant to be pathogenic, monogenic disease would have to be seen in one in 5000 population, which is far more common than the estimated prevalence of hyperthyroidism due to activating TSHR mutations (~<1:15 000 children in Denmark). 12,20 None of the previously published pathogenic activating TSHR mutations are seen in gnomAD.
Furthermore, the proband with this variant in our study was diagnosed at 60 years of age and had no family history of thyrotoxicosis. She also remitted after 18 months of antithyroid drug treatment which is unlikely if she had activating TSHR gene mutation.

| D ISCUSS I ON
Our study identified that the systematic assessment for activating TSHR mutations in adult patients with hyperthyroidism with diffuse uptake on scintigraphy, negative TRAb and absence of extrathyroidal manifestations of Graves' disease (such as thyroid-associated ophthalmopathy or dermopathy) did not identify a single case. Our study, therefore, is unable to support the routine genetic testing for TSHR mutations in these patients.
This is the largest study in adult Caucasian patients with hyperthyroidism with diffuse goitre and lacking overt autoimmunity, and we did not find any individuals with a pathogenic variant in theTSHR gene. Our study results differ from a previous study in adult Japanese hyperthyroid patients with diffuse goitre and without TRAb (n = 89). 13 This study found activating TSHR mutations in four patients, giving a prevalence of 4.5%. This discrepancy in the results may be due to different populations characteristics; for example, our study participants were older, median age 52 vs 40 years. 13 The high frequency in the Japanese population may be due to founder effects as seen in other monogenic diseases in this population. 21 The negative results in our study may also be due to a type II error (false negative), although we were powered to detect a 4.5% frequency with 95% confidence. This suggests that the accurate assessment of prevalence of activating TSHR muta- We also did not take the treatment history of patients (eg, relapse after antithyroid drug treatment) into consideration when selecting potential participants as we wanted to establish the utility of genetic testing at the time of diagnosis during routine clinical care.
In conclusion, our systematic assessments of adult Caucasian patients with hyperthyroidism showed that activating TSHR gene mutations are a rare cause of nonautoimmune hyperthyroidism. Our results do not support the routine genetic testing for adult patients with hyperthyroidism showing diffuse uptake on scintigraphy but lacking autoimmune features of Graves' disease.

ACK N OWLED G EM ENTS
We would like to acknowledge Bradley Harman and Rebecca Ward for undertaking Sanger sequencing.

CO N FLI C T O F I NTE R E S T
The authors report no conflict of interests that could be perceived as prejudicing the impartiality of the research reported.

AUTH O R S' CO NTR I B UTI O N S
KAP and BV designed the study; wrote the first draft of the manuscript, which was modified by all authors; and are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of