De Novo SOX10 Nonsense Mutation in a Patient with Kallmann Syndrome, Deafness, Iris Hypopigmentation, and Hyperthyroidism

Available online at www.annclinlabsci.org 248 Annals of Clinical & Laboratory Science, vol. 48, no. 2, 2018 De novo SOX10 Nonsense Mutation in a Patient with Kallmann Syndrome, Deafness, Iris Hypopigmentation, and Hyperthyroidism

Fang Wang*, Shaoli Zhao*, Yanhong Xie, Wenjun Yang, and Zhaohui Mo

The Endocrinology Department of the Third Xiangya Hospital, Central South University, Changsha, Hunan Province, China

Abstract. Kallmann syndrome (KS) is a clinically and genetically heterogeneous disorder characterized by hypogonadotropic hypogonadism and olfactory dysfunction. Recently, mutations in SOX10, a well-known causative gene of Waardenburg syndrome (WS), have been identified in a few KS patients with additional developmental defects including hearing loss. However, the understanding of SOX10 mutation associates with KS and other clinical consequences remains fragmentary. A 30-year-old Chinese male patient presented with no pubertal sex development when he was at the age of twelve years. Additionally, he showed anosmia, sensory deafness, and blue irises. Last year, he developed clinical symptoms of hyperthyroidism with a fast heartbeat, heat intolerance and weight loss. Blood examinations revealed low levels of FSH, LH, and testosterone. Thyroid function showed high levels of FT3, FT4 and extremely low level of TSH. Molecular analysis detected a de novo (c.565G>T/p.E189X) mutation in SOX10, which has previously been reported in a patient with WS4 (WS with Hirschsprung). The mutation was predicted to be probably damaging. These results highlight the significance of SOX10 haploinsufficiency as a genetic cause of KS. Importantly, our result implies that the same SOX10 mutation can underlie both typical KS and WS, while the correlation between SOX10 and hyperthyroidism still needs to be clarified in the future.

Key words: SOX10, Kallmann syndrome, Waardenburg syndrome, hypogonadotropic hypogonadism, Hyperthyroidism.

Introduction due to a de novo SOX10 nonsense mutation [6]. Furthermore, Suzuki et al. described SOX10 muta- Kallmann syndrome (KS), a combination of con- tion in a patient with KS, hearing loss and iris hy- genital hypogonadotropic hypogonadism and an- popigmentation in 2015 [7]. These results indicat- osmia, is a clinically and genetically heterogeneous ed a significant role of SOX10 mutation underlying condition [1]. More than 20 genes have been con- KS with deafness. Since SOX10 is a well-known firmed to be related to KS, although mutations in causative gene of WS, previous SOX10 mutation these genes account for only a small fraction of the analyses have primarily focused on patients with etiology KS (approximately 30%) [2-4]. KS can be WS4 or WS2 [8-10]. However, the genetic links associated with several additional developmental between KS and WS have not been established and defects such as hearing loss. In 2013, Pingault et al. the detailed clinical manifestations of SOX10 mu- identified a high prevalence (about 38%) of SOX10 tations remain to be fully clarified. Here, we report mutations in patients with KS and deafness [5]. In a patient with KS, hearing impairment, hypopig- addition, he described an important founding that mented iris, and hyperthyroidism, who carried a de genetic knockout of SOX10 in murine fetuses dis- novo SOX10 nonsense mutation. rupted the migration of GnRH cells, the deficiency of which account for KS. In 2014, Vaaralahti et al. Case Report reported a male patient with KS and hearing loss A 30-year-old male patient was referred to us for delayed *These authors contributed equally to this work. Address puberty and weight loss. He was born to nonconsan- correspondence to Zhaohui Mo, The Endocrinology Department of the Third Xiangya Hospital, Central South University, 138# Tong guineous Chinese parents after a normal pregnancy. Zipo Road, Changsha, Hunan Province, China 410013; phone: Shortly after birth, he presented with hypopigmented 13875835371; email address: [email protected] irides (Figure 1). And he reported anosmia and hearing

0091-7370/18/0200-248. © 2018 by the Association of Clinical Scientists, Inc. SOX10 mutation in KS, WS, hyperthyroidism 249 GnRH stimulation. Thyroid hormones suggested hyperthyroidism with high levels of FT3(45.95pmol/L; normal range: 3.1- 6.8), FT4 (>100pmol/L; normal range: 10.3-22.65), Figure 1. Depigmentation of the irides. TRAb (4.38IU/L; normal range: 0-1.75), TPO (385IU/ml; normal range: 0-34), TGA (979.8IU/ml; normal range: 0-115) and low level of TSH (<0.005uIU/ml; normal range: 0.27-4.2). In addition, the anti- mitochondrial antibody (AMA) in the serum of the patient is positive. The blood values of other hormones such as ACTH and cortisol were grossly normal. Figure 2. T2-weighted brain magnetic resonance imaging. No abnormalities were noted in the hypo- thalamus or pituitary. Auditory brainstem response indicated bilateral sensorineural deafness. Brain magnetic resonance imaging (MRI) showed no abnormalities in the hy- pothalamus or pituitary (Figure 2). These findings suggested that the patient had signifi- cantly delayed pubertal maturation. However, his parents, younger brother and younger sister were phenotypi- Figure 3. The de novo mutation in SOX10 gene (c.565G>T). cally normal. loss. In addition, he got symptoms of hyperthyroidism such as Methods. After obtaining written informed consent, genom- accelerated heartbeat, heat intolerance and sudden weight loss ic DNA was extracted from peripheral blood samples ob- last year. He had normal mental development. Initial physical tained from the patient and his family members using examination showed a lack of pubertal signs such as micropenis QiaAmp Blood Kit (Qiagen). In view of his sensorineural (1.5cm) with unpalpable testes, breast, and pubic hair was deafness and iris hypopigmentation, we considered this KS Tanner stage 1. His height was 158cm. Blood examinations re- patient also had WS which can be caused by SOX10 muta- vealed the testosterone level (10.37ng/dl; normal range: 249- tion. Thus, we carried out mutation screening for the coding 836), gonadotropin levels including FSH (0.62mIU/ml; nor- region of SOX10. Six sets of primers (sequences will be pro- mal range: 1.5-12.4) and LH (0.82mIU/ml; normal range: vided upon requests) were used to amplify SOX10 gene frag- 1.7-8.6) were very low, with minor and delayed response to ments covering exons 1 to 5 through polymerase chain 250 Annals of Clinical & Laboratory Science, vol. 48, no. 2, 2018 reaction (PCR). Sequences of the PCR products were obtained using the ABI 3100 Genetic Analyzer (ABI, Foster City, CA). Mutations were confirmed by sequenc- ing duplicate PCR templates from separate reactions. Several databases were used to predict the effect of the sequence variants on the function of the protein, including polyphen2 (polymorphism phenotyping, http://genetics.bwh.harvard.edu/pph2/), SIFT (Sorting Intolerant From Tolerant, http://sift.bii.astar.edu.sg/) and MutationTaster (www.mutationtaster.org).

Results

By sanger sequence, a nonsense mutation (c.565G>T/p.E189X) in SOX10 was identified. The 2nd exon in the SOX10 coding region has a heterozygous mutation of replacement of the 565th G by T, resulting in replacement of the 189th glu- tamic acid codon by a stop codon and possible ter- Figure 4. The proband’s family pedigree. minating the translation in advance (Figure 3). We inquire several databases and references and find Waardenburg syndrome. Additionally, our patient this mutation has been reported in a WS4 patient met the diagnosis criteria of WS1 with W index 2.2 in 1998, who carried Hirschsprung disease but (>1.95). W index was calculated by a formula without the description of KS. This mutation was which based on inner canthus, pupillary distance confirmed as de novo as the reason that no SOX10 and outer canthus [10]. The different phenotype mutation could be detected in her parents and oth- between the previously reported child and our pa- er family members (Figure 4). Meanwhile, the mu- tient suggest the incomplete penetrance and vari- tation was not presented in dbSNP and Exome able expressivity of clinical features resulting from Variant Server databases. Furthermore, no patho- SOX10 abnormalities. To date, 11 KS patients have genic mutations were identified in any of the other been described with SOX10 mutations, in which 9 KS responsible genes. All these results indicate that patients carried deafness while 5 patients showed this mutation was probably damaging. hypopigmentation and no Hirschsprung disease was observed [4-7]. Our results, together with these Discussion previous findings, indicate the importance of SOX10 haploinsufficiency as one of the genetic Herein, we report a case of KS patient associated cause of KS with WS-characteristic clinical with WS and hyperthyroidism, who carried a de features. novo (c.565G>T/p.E189X) mutation of SOX10. The same mutation of SOX10 has previously been The SOX10 protein belongs to SOX gene super- reported in a child with WS and Hirschsprung dis- family with a highly conserved center of high mo- ease, in whom the presence or absence of hypogo- bility group (HMG domain). It is a key transcrip- nadotropic hypogonadism and anosmia has not tion factor in the development and migration of been described and yet to be determined [11]. neural crest, which can differentiate into melano- However, Hirschsprung disease was absent in our cytes, olfactory ensheathing cells and enteric gan- patient. To our knowledge, this is the first case of glia neurons [12-14]. In this case, the SOX10 pro- KS associated with WS and hyperthyroidism in a tein was truncated by a stop codon (E189X) result patient carried this SOX10 mutation. Our patient from SOX10 mutation, making dysfunction of me- manifested hypogonadotropic hypogonadism with lanocytes and olfactory ensheathing cells (Figure anosmia, which was characteristic of Kallmann 5). On one hand, dysfunction of melanocytes can syndrome. Interestingly, he presented with bilateral result in defects of melanin synthesis, which is im- sensorineural deafness and hypopigmented irides portant for the development of the inner ear, lead- simultaneously, which can be diagnosed with ing to congenital hearing impairment and iris SOX10 mutation in KS, WS, hyperthyroidism 251

Figure 5. Schematic diagram illustrating the proposed effect of SOX10 mutation leading to KS and WS. heterochromia. On the other hand, defects in the as insulin-dependent diabetes mellitus and thyroid development of olfactory ensheathing cells can lead disease [18-21]. Hedstrand et al. identified SOX10 to anosmia and hypogonadotropic hypogonadism, as an autoantigen in patients with autoimmune since olfactory ensheathing cells are required for polyendocrine syndrome type I (APS I), which is gonadotropin-releasing hormone neuron migra- known to have high titer autoantibodies directed tion. The reason why this patient did not experi- against various tissue-specific antigens [22]. These ence Hirschsprung disease can be explained by the discoveries suggested that SOX10 has an important notion that SOX10 haploinsufficiency is usually role in the development of autoimmune disease. associated with a wide range of penetrance and Therefore, we hypothesized that SOX10 may be also expressivity[15]. associated with hyperthyroidism, possibly through immune-mediated mechanisms. Further studies are In this case, besides KS and WS, the patient also needed to investigate if SOX10 is one of the patho- had hyperthyroidism caused by Graves' disease genic factors underlying the development of hyper- with elevated thyroid autoantibodies including thyroidism and/or other autoimmune disorders. TRAb, TPO, and TGA. In addition, the anti-mitochondrial antibody (AMA) in the serum of the pa- Conclusions. We identified a SOX10 mutation in a tient is also positive, indicating the presence of or patient with clinically diagnosed KS, WS, and hy- the potential to develop autoimmune disease. perthyroidism. This case provides further evidence However, this condition occurred just one year be- that SOX10 could be the same genetic cause of KS fore, indicating it was not a congenital disorder but and WS, which possibly share a common regulatory acquired after. This analysis prompted us to con- pathway in some crucial phase of neural crest devel- sider the role of SOX10 in autoimmune disease. To opment. Therefore, additional phenotypic cues date, there are no reports covering the relationship should be searched and thorough clinical evalua- between SOX10 and hyperthyroidism. However, tions should be performed for KS or/and WS pa- recent studies indeed suggested SOX10 was associ- tient. Further studies are needed to clarify the char- ated with some autoimmune disorders such as vit- acteristics of SOX10 abnormalities and the links to iligo and melanoma, in which both humoral and autoimmune disorders such as hyperthyroidism. cellular mechanisms are believed to be involved [16-17]. Blokzijl et al. described serum levels of Acknowledgements SOX10 in vitiligo and melanoma patients were el- This study was supported by the National Natural Science evated compared to healthy controls, indicating on- Foundation of China (No.81400831); The National going destruction of SOX10-containing cells Natural Science Foundation of China (No.81670769); The New Xiangya Talent Project of the Third Xiangya through immune-mediated mechanisms [17]. Hospital of Central South University (No.JY201718); Interestingly, vitiligo has been demonstrated to be The New Xiangya Talent Project of the Third Xiangya associated with various autoimmune disorders such Hospital of Central South University (20150307). 252 Annals of Clinical & Laboratory Science, vol. 48, no. 2, 2018

References 10. Ma J, Zhang TS, Lin K, Sun H, Jiang HC, Yang YL, Low F, Gao YQ, Ruan B. Waardenburg syndrome type II in a Chinese 1. Trarbach EB, Silveira LG, Latronico AC. Genetic insights patient caused by a novel nonsense mutation in the SOX10 into human isolated gonadotropin deficiency. Pituitary 2007; gene. Int J Pediatr Otorhinolaryngol 2016; 85:56-61. 10:381-391. 11. Pingault V, Bondurand N, Kuhlbrodt K, Goerich DE, Préhu 2. Layman LC. The genetic basis of female reproductive disor- MO, Puliti A, Herbarth B, Hermans-Borgmeyer I, Legius E, ders: etiology and clinical testing. Mol Cell Endocrinol 2013; Matthijs G, Amiel J, Lyonnet S, Ceccherini I, Romeo G, Smith 370:138-148. JC, Read AP, Wegner M, Goossens M. SOX10 mutations in 3. Miraoui H, Dwyer AA, Sykiotis GP, Plummer L, Chung W, patients with Waardenburg-Hirschsprung disease. Nat Genet Feng B, Beenken A, Clarke J, Pers TH, Dworzynski P, Keefe 1998; 18:171-173. K, Niedziela M, Raivio T, Crowley WF Jr, Seminara SB, 12. Barraud P, St John JA, Stolt CC, Wegner M, Baker CV. Quinton R, Hughes VA, Kumanov P, Young J, Yialamas Olfactory ensheathing glia are required for embryonic olfacto- MA, Hall JE, Van Vliet G, Chanoine JP, Rubenstein J, ry axon targeting and the migration of gonadotropin-releasing Mohammadi M, Tsai PS, Sidis Y, Lage K, Pitteloud N. hormone neurons. Biol Open 2013; 2:750-759. Mutations in FGF17, IL17RD, DUSP6, SPRY4, and FLRT3 13. Hou L, Arnheiter H, Pavan WJ. Interspecies difference in the are identified in individuals with congenital hypogonado- regulation of melanocyte development by SOX10 and MITF. tropic hypogonadism. Am J Hum Genet 2013; 92:725-743. Proc Natl Acad Sci USA 2006; 103:9081-9085. 4. Izumi Y, Musha I, Suzuki E, Iso M, Jinno T, Horikawa R, 14. Kuhlbrodt K, Herbarth B, Sock E, Hermans-Borgmeyer I, Amemiya S, Ogata T, Fukami M, Ohtake A. Wegner M. SOX10, a novel transcriptional modulator in glial Hypogonadotropic hypogonadism in a female patient previ- cells. J Neurosci 1998; 18:237-250. ously diagnosed as having waardenburg syndrome due to a 15. Fisher E, Scambler P. Human haploinsufficiency-one for sor- sox10 mutation. Endocrine 2015; 49:553-556. row, two for joy. Nat Genet 1994; 7:5-7. 5. Pingault V, Bodereau V, Baral V, Marcos S, Watanabe Y, 16. Passeron T, Ortonne JP. Physiopathology and genetics of vitil- Chaoui A, Fouveaut C, Leroy C, Vérier-Mine O, Francannet igo.J Autoimmun 2005; 25:63-68. C, Dupin-Deguine D, Archambeaud F, Kurtz FJ, Young J, 17. Blokzijl A, Chen LE, Gustafsdottir SM, Vuu J, Ullenhag G. Bertherat J, Marlin S, Goossens M, Hardelin JP, Dodé C, Elevated levels of SOX10 in serum from vitiligo and melanoma Bondurand N. Loss-of-function mutations in SOX10 cause patients, analyzed by proximity ligation assay. PLoS One 2016; Kallmann syndrome with deafness. Am J Hum Genet 2013; 11:e0154214. 92:707-724. 18. Betterle C, Caretto A, De Zio A, Pedini B, Veller-Fornasa C, 6. Vaaralahti K, TommiskaJ, Tillmann V, LiivakN, Känsäkoski Cecchetto A, Accordi F, Peserico A. Incidence and significance J, Laitinen EM, Raivio T. De novo SOX10 nonsense muta- of Organ-specific autoimmune disorders (clinical, latent or tion in a patient with Kallmann syndrome and hearing loss. only autoantibodies) in patients with vitiligo. Dermatologica Pediatr Res 2014; 76:115-116. 1985; 171:419-423. 7. Suzuki E, Izumi Y, Chiba Y, Horikawa R, Matsubara Y, 19. Gould IM, Gray RS, Urbaniak SJ, Elton RA, Duncan LJ. Tanaka M, Ogata T, Fukami M, Naiki Y. Loss-of-function Vitiligo in diabetes mellitus. Br J Dermatol 1985; SOX10 mutation in a patient with Kallmann syndrome, 113:153-155. hearing loss, and iris hypopigmentation. Horm Res Paediatr 20. Cunliffe WJ, Hall R, Newell DJ, Stevenson CJ. Vitiligo, thy- 2015; 84:212-216. roid disease, and autoimmunity. Br J Dermatol 1968; 8. Pingault V, Ente D, Dastot-Le Moal F, Goossens M, Marlin 80:135-139. S, Bondurand N. Review and update of mutations causing 21. Schallreuter KU, Lemke R, Brandt O, Schwartz R, Westhofen Waardenburg syndrome. Hum Mutat 2010; 31:391-406. M. Vitiligo and other diseases: coexistence or true association? 9. Bondurand N, Dastot-Le Moal F, Stanchina L, Collot N, Hamburg study on 321 patients. Dermatology 1994; Baral V, Marlin S, Attie-Bitach T, Giurgea I, Skopinski L, 188:269-275. Reardon W, Toutain A, Sarda P, Echaieb A, Lackmy-Port-Lis 22. Hedstrand H, Ekwall O, Olsson MJ, Landgren E, Kemp EH. M, Touraine R, Amiel J, Goossens M, Pingault V. Deletions The transcription factors SOX9 and SOX10 are vitiligo autoan- at the SOX10 gene locus cause Waardenburg syndrome types tigens in autoimmune polyendocrine syndrome type I. J Biol 2 and 4. Am J Hum Genet 2007; 81:1169-1185. Chem 2001; 276:35390-35395.