A NOVEL COMPOUND HETEROZYGOUS VARIANT OF SLC12A3 GENE IN A PEDIGREE WITH GITELMAN SYNDROME COEXISTED WITH THYROID DYSFUNCTION

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1 ENDOCRINE PRACTICE Rapid Electronic Article in Press Rapid Electronic Articles in Press are preprinted manuscripts that have been reviewed and accepted for publication, but have yet to be edited, typeset and finalized. This version of the manuscript will be replaced with the final, published version after it has been published in the print edition of the journal. The final, published version may differ from this proof. Original Article EP A NOVEL COMPOUND HETEROZYGOUS VARIANT OF SLC12A3 GENE IN A PEDIGREE WITH GITELMAN SYNDROME COEXISTED WITH THYROID DYSFUNCTION Simo Liu MD #, Jing Ke MD, PhD #, Baoyu Zhang MD, Caiguo Yu MD, Yingmei Feng MD, PhD, Dong Zhao MD From: Beijing Key Laboratory of Diabetes Mellitus Prevention and Research, Department of Endocrinology, Beijing Luhe Hospital, Capital Medical University, Beijing , China # The authors share equal contribution to the study Running title: Gitelman syndrome and thyroid dysfunction Corresponding address: Dong Zhao MD or Yingmei Feng, MD, PhD Beijing Key Laboratory of Diabetes Mellitus Prevention and Research, Department of Endocrinology, Beijing Luhe Hospital, Capital Medical University, Beijing , China address: zdoc66@126.com yingmeif13@ccmu.edu.cn; yingmeif13@sina.com

2 Abstract Objective: Gitelman syndrome (GS) is known as an autosomal recessive disorder characterized by salt wasting and hypokalemia resulting from mutations in the SLC12A3 (solute carrier family 12 member 3) gene, which codes the thiazide-sensitive Na-Cl cotransporter. To date, more than 488 mutations of the SLC12A3 gene have been discovered in patients with GS. In this study, we reported a GS pedigree complicated by thyroid diseases or thyroid dysfunction. Methods: Sanger sequencing and next generation sequencing analysis were performed to determine the SLC12A3 gene mutations in a GS pedigree including the 16-year old male patient with GS and his family members within three generations. Chemiluminescence immunoassays were used to detect TSH, FT3, TT3, FT4, TT4, TGAb and TPOAb concentrations. Results: Genetic analysis of the SLC12A3 gene identified two mutations in the 16-year old male patient with GS concomitant with Graves disease (GD) and his younger sister accompanied by abnormal thyroid function. Additionally, one mutation site(c.1456g>a) in SLC12A3 gene was found in his father, paternal uncle and elder female cousin, who were complicated by subclinical hypothyroidism or autoantibody against thyroid. The other mutation site (c.2102_2107 delacaaga) in SLC12A3 gene, a novel mutated variant of SLC12A3 gene, was carried by his mother and maternal grandfather. Conclusions: Two mutation sites were documented in the pedigree with GS, and one has not been reported before. Moreover, we found mutation at nucleotide c.1456 G>A in SCL12A3 gene which may affect thyroid function. However, further studies are particularly needed to explore the underlying molecular mechanisms. Key words: Gitelman syndrome, SLC12A3 gene, thyroid function

3 Abbreviations: FT3 = free triiodothyronine; FT4 = free tetraiodothyronine; GS = Gitelman Syndrome; GD = Graves disease; SLC12A3 = solute carrier family 12 member 3; TGAb = thyroglobulin antibody; TT3 = total triiodothyronine; TT4 = total tetraiodothyronine; TPOAb = thyroid peroxidase antibody; TPP = thyrotoxic periodic paralysis; TSH = thyroid-stimulating hormone. Introduction Gitelman syndrome (GS) is an autosomal recessive disorder characterized by hypokalemia, metabolic alkalosis, normal blood pressure, hyperreninemic hyperaldosteronism, hypomagnesaemia and hypocalciuria (1). GS is generated by mutations in the gene SLC12A3 encoding the thiazide-sensitive Na-Cl cotransporter (2). The human SLC12A3 gene, located on chromosome 16, consists of 26 exons and encodes a protein that containing 12 putative transmembrane domains (3). To date, more than 488 kinds of SLC12A3 gene mutations have been discovered including missense, shear, nonsense, frame shift, insertion or deletions and more (4). Graves disease (GD) is an autoimmune thyroid disease involving an excessive amount of the thyroid hormone and leads to the increased excitability of the nervous, circulatory, digestive and imbalance of electrolytes including calcium and phosphorous metabolism disorders and low blood potassium. Several case reports have shown that GS and GD coexisted in one patient (1, 5), suggesting there may be some connections between them. It has been reported that thyroid status could affect electrolyte balance in the kidney. Disturbances of thyroid function are accompanied by widespread alterations in renal hemodynamics and tubular handling of electrolytes (6, 7). It has been reported that the increased electrolyte excretion in the kidney due to excess thyroid hormone may have exacerbated the clinical features of GS (7). Our study documented one case of GS concomitant with GD, and we summarized the thyroid function and SLC12A3 gene status in the GS pedigree as well.

4 Methods Samples collection A peripheral blood sample (2 ml) were collected into tubes containing 50 mmol/l EDTA-2Na and genomic DNAs were extracted and examined by Macro & micro test (Beijing, China). Screening for mutations in SLC12A3 To define and confirm the SLC12A3 gene status of the patient (proband III7) and this pedigree, sanger sequencing and next generation sequencing analysis were performed. All of the studies were accomplished in the Macro & micro test (Beijing, China). Laboratory testing Chemiluminescence immunoassays were used to detect thyroid-stimulating hormone (TSH), total triiodothyronine (TT3), total tetraiodothyronine (TT4), free triiodothyronine (FT3), free tetraiodothyronine (FT4), thyroglobulin antibody (TGAb) and thyroid peroxidase antibody (TPOAb) [Cobas 6000 analyzer, Roche Diagnostics, Shanghai] concentrations. Results Proband III7 A 16-year-old teenager was admitted to the Endocrinology department of our hospital for recurrent pain in lower extremity and fatigue. Hypokalemia was diagnosed based on a serum potassium level of mmol/l in the past one year prior to the hospitalization. He has been treated for GD for half a year. No similar signs and symptoms were noted in his family members. On admission, proband III7 complained of severe muscle weakness and paresthesia. The results of standard physical examinations were unremarkable besides enlarged thyroid gland and heart rate value of up to 110 beats per minute. The biochemical analysis showed metabolic alkalosis with hyocalciuria (0.33 mmol/24 h, normal reference value mmol/24 h) and nearly normal serum calcium (2.54 mmol/l, normal reference value mmol/l). The level of serum magnesium was decreased to 0.4

5 mmol/l (normal reference value mmol/l). In clinostatism test, the level of renin and aldosterone were pg/ml (normal reference value 4-24 pg/ml) and pg/ml (normal reference value pg/ml). While in stand test, the level of renin and aldosterone were pg/ml (normal reference value 4-38 pg/ml) and pg/ml (normal reference value pg/ml). His TSH level was less than 0.00 uiu/ml (normal reference value uiu/ml), FT4 level was 5.74 ng/dl (normal reference value ng/dl) and FT3 level was pg/ml (normal reference value pg/ml). TGAb was 231 U/mL (normal reference value <115 U/mL), TPOAb was U/mL (normal reference value <34 U/mL), and TSH receptor antibody level increased to IU/L (normal reference value <1.75 IU/L). Ultrasound results showed the diffuse enlargement of thyroid, which was one of the signs of GD. CT scan of adrenal glands were normal. GD complicated with thyrotoxic periodic paralysis (TPP) was diagnosed according to the laboratory tests. Methimazole was given orally at 30 mg/day, oral potassium chloride release tablets and potassium citrate granules were given administered. The serum potassium concentration fluctuated around the level of 2.98 mmol/l after one week s treatment. We corrected our diagnosis as GD combined with GS after clinical and performed genetic examinations. Genetic detection of peripheral blood specimens found that SLC12A3 gene mutation, in which exon 12 in 1456 nucleotides with a G to A mutation, and exon 17 in 2102_2107 nucleotides with deletion. Oral potassium chloride release tablets (3 g/day), spironolactone (60 mg/day) and potassium citrate granules (4.35 g/day), potassium magnesium aspartate (20 ml/day) were prescribed. The serum potassium and magnesium level rose to 3.77 mmol/l and 0.64 mmol/l respectively, while normalization of thyroid function was noted and methimazole was given orally at 5 mg/day after four weeks. Gene mutation analysis SLC12A3 gene mutation analysis and relevant laboratory testing were performed in proband III7 and his relatives within three generations. Similar apparent clinical symptoms were not noted in all other family members. Informed consent was obtained from each individual according to a protocol approved by the human studies committee of Beijing

6 Luhe hospital. Proband III7 was found to be a compound heterozygote with a single-base substitution at nucleotide c.1456 G>A in exon 12 and a deletion at nucleotide c.2102_2107 (delacaaga) in exon 17. Familial linkage analyses confirmed that c.1456g>a was the paternal allele and c.2102_2107 was the maternal allele (Figure 1). Laboratory results of other family members Patient III6, the sister of proband III7, has the same genotype as proband III7, while no obvious symptom presented. Patient III6 has nearly normal levels of serum potassium, magnesium, and calcium. Thyroid function showed T3 level was 2.39 ng/ml (normal reference value pg/ml), FT3 level was 5.65 pg/ml, normal reference value ng/dl), TSH level was 6.82 uiu/ml (normal reference value uiu/ml). At the follow-up 6 months after entry, we observed fatigue and serum hypokalemia (2.99 mmol/l) and hypocalciuria. In addition, the biochemical analysis showed hyocalciuria (0.19 mmol/24 h, normal reference value mmol/24h). Based on the clinical and biochemical results, GS could be diagnosed in the patient III6. Interestingly, family members carrying mutation site at nucleotide c.1456 G>A in exon 12 have mild abnormal thyroid function, including increased TSH or autoantibody against thyroid. Whereas Proband III7 and patient III6 have more severe thyroid dysfunction (Table 1). According to these results, we speculated that mutation at nucleotide c.1456 G>A in SCL12A3 gene may affect thyroid function. Discussion The case of hypokalemia in a teenage boy, previously diagnosed and treated as GD and then re-diagnosed as GS concomitant with GD following genetic counseling, not only identifies c.2102_2107 (delacaaga) in exon 17 as a novel variant of SLC12A3 gene, but also suggests mutation at nucleotide c.1456 G>A of SLC12A3 gene may affect thyroid function. It is generally acknowledged that an interaction exists between functions of thyroid gland and the kidney (8). Thyroid dysfunction affects renal physiology and development, whereas kidney disease could result in thyroid dysfunction. Hyperthyroidism could give

7 rise to increased GFR as well as activation of renin-angiotensin-aldosterone system (9). It has been shown that patients with thyroid disease may tend to develop symptoms of GS (10). It is possible that the symptoms of GS appear prominent when exposed to pathophysiological force to induce electrolyte imbalance such as hyperthyroidism. Numerous kidney diseases such as chronic kidney disease and glomerulonephritides have been demonstrated to be associated with abnormal thyroid function including reduced T3, lower serum T4 or status of Hashimoto s thyroiditis (11-13). Several cases coexist with GS and GD have been reported. Zha et al (5) have reported a 14-year-old young girl with GD and GS, and exon 6 in 791 nucleotides with a G to C mutation was found. Then three Japanese patients with genetically confirmed GS complicated by GD were diagnosed and reported (1). In our study, the proband was diagnosed with GS and GD. Furthermore, we performed SLC12A3 gene mutation analysis in three successive generations and found that coexistence with GS and abnormal thyroid function were found in patient III6. Besides, we found that family members carrying mutation at nucleotide c.1456 G>A in SCL12A3 gene almost have abnormal thyroid function, including elevated T3, FT3, TSH or thyroid autoantibodies. In the patient s sister, the abnormal thyroid function presented with elevated T3, FT3 and TSH, which is more severe than other carriers of the gene mutation. It has been early reported that homozygous mutation at c.1456 G>A in exon 12 contributed to the development of GS, while no data of thyroid function data presented (3). Family linkage study in the present case exhibited that carriers with mutation at nucleotide c.1456 G>A in exon 12 almost have abnormal thyroid function whereas those with two mutation sites were accompanied by more severe thyroid dysfunction, suggesting the possible association between the gene mutation and thyroid function. Evidence showing that thyroid status could affect calcium and magnesium metabolism (13), which may explain that prominent symptoms of GS in the proband and only mild symptom even no abnormity earlier in patient III6. Although there exist several cases of patients with GS complicated by GD, the specific mechanism and whether GS could have effect on thyroid function remained unclear. Further studies are needed to illuminate the possible underlying mechanism of the association of GS with thyroid disease.

8 On the other hand, in clinical work, GS may be easily misdiagnosed as hypokalemic periodic paralysis, such as thyrotoxic periodic paralysis of familial periodic paralysis due to acute shift of K + into cells. Thyrotoxicosis is the most common cause of hypokalemic periodic paralysis in GD, especially in Asian men (14). As the prevalence of GS is not necessarily low, and clinicians need to be aware of GS as one possible cause of hypokalemia in patients with GD. Conclusions In summary, this study identified a novel pathogenic variant of SLC12A3 in a GS pedigree and mutation at nucleotide c.1456 G>A in SCL12A3 gene, which appears to be associated with thyroid dysfunction. However, further studies are still needed to clarify and illuminate the underlying mechanism. In the future, we will design an animal model with gene mutation to illuminate the association between the mutation and thyroid function. Acknowledgements The authors would like to express sincere thanks to the patient for sharing her clinical data. Sources of funding The authors declare no conflicts of interest. Conflict of interest None to declare. Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was approved by Ethics Committee of Beijing Luhe Hospital. This article does not contain any studies with animals performed by any of the authors.

9 Informed consent Informed consent was obtained from all individual participants included in the study. Reference 1. Mizokami T, Hishinuma A, Kogai T, et al. Graves' disease and Gitelman syndrome. Clinical endocrinology. 2016;84: Parmar MS, Bhimji SS. Gitelman Syndrome. StatPearls. Treasure Island FL: StatPearls Publishing LLC., Simon DB, Nelson-Williams C, Bia MJ, et al. Gitelman's variant of Bartter's syndrome, inherited hypokalaemic alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl cotransporter. Nature genetics. 1996;12: Lu Q, Zhang Y, Song C, et al. A novel SLC12A3 gene homozygous mutation of Gitelman syndrome in an Asian pedigree and literature review. Journal of endocrinological investigation. 2016;39: Zha B, Zheng P, Liu J, Huang X. Coexistence of Graves' Disease in a 14-year-old young girl with Gitelman Syndrome. Clinical endocrinology. 2015;83: Jones JE, Desper PC, Shane SR, Flink EB. Magnesium metabolism in hyperthyroidism and hypothyroidism. The Journal of clinical investigation. 1966;45: McCaffrey C, Quamme GA. Effects of thyroid status on renal calcium and magnesium handling. Canadian journal of comparative medicine: Revue canadienne de medecine comparee. 1984;48: Kaptein EM. Thyroid function in renal failure. Contributions to nephrology. 1986;50: den Hollander JG, Wulkan RW, Mantel MJ, Berghout A. Correlation between severity of thyroid dysfunction and renal function. Clinical endocrinology. 2005;62:

10 10. Aoi N, Nakayama T, Tahira Y, et al. Two novel genotypes of the thiazide-sensitive Na-Cl cotransporter (SLC12A3) gene in patients with Gitelman's syndrome. Endocrine. 2007;31: Akikusa B, Kondo Y, Iemoto Y, Iesato K, Wakashin M. Hashimoto's thyroiditis and membranous nephropathy developed in progressive systemic sclerosis (PSS). American journal of clinical pathology. 1984;81: Wiederkehr MR, Kalogiros J, Krapf R. Correction of metabolic acidosis improves thyroid and growth hormone axes in haemodialysis patients. Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association. 2004;19: Basu G, Mohapatra A. Interactions between thyroid disorders and kidney disease. Indian journal of endocrinology and metabolism. 2012;16: Molin CZD, Trevisol DJ. Persistent severe hypokalemia: Gitelman syndrome and differential diagnosis. Jornal brasileiro de nefrologia : 'orgao oficial de Sociedades Brasileira e Latino- Americana de Nefrologia. 2017;39:

11 Figures Figure 1. Sequence analysis of the SLC12A3 gene. A heterozygous transition (G to A) at nucleotide c.1456 in exon 12 and a deletion at nucleotide c.2102_2107 (delacaaga) in exon 17 were indicated by a red arrow

12 Figure 2. SLC12A3 gene mutation analysis in three consecutive generations of the family. Arrow denotes the proband. Individuals affected by gene mutation (c.1456 G>A) in exon 12 were indicated with half-filled black symbols. Individuals subjects affected by gene mutation (c.2102_2107 (delacaaga) in exon 17 are indicated with half-filled slash symbols. Table 1. Thyroid function and gene mutation site of patients and carriers FT3, free triiodothyronine, FT4, free thyroxine, TT3, total triiodothyronine, TT4, total thyroxine, TRAb, TSH receptor antibody. TGAb, thyroglobulin antibody, TPOAb, thyroid peroxidase antibody. Bold fonts for previously unreported mutation. Reference range: T3, ng/ml, T4, ug/dl, FT3, pg/ml, FT4, ng/dl, TSH uiu/ml, TGAb, <115 U/ml, TPOAb, <34 U/ml, A-TSHR <1.75 IU/L.