Monogenic Hashimoto thyroiditis associated with a

Journal of Autoimmunity 86 (2018) 116e119

Contents lists available at ScienceDirect

Journal of Autoimmunity journal homepage: www.elsevier.com/locate/jautimm

Monogenic Hashimoto thyroiditis associated with a variant in the thyroglobulin (TG) gene Mindy S. Lo a, *, Meghan Towne b, d, Grace E. VanNoy b, d, Catherine A. Brownstein b, d, Andrew A. Lane e, Talal A. Chatila a, Pankaj B. Agrawal b, c, d a

Divisions of Immunology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, United States Genetics and Genomics, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, United States Division of Newborn Medicine, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, United States d The Manton Center for Orphan Disease Research, Boston Children's Hospital and Harvard Medical School, Boston, MA, United States e Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, United States b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 August 2017 Received in revised form 8 September 2017 Accepted 11 September 2017 Available online 21 September 2017

Background: Risk of autoimmune thyroid disease (AITD) is strongly heritable. Multiple genes confer increased risk for AITD, but a monogenic origin has not yet been described. We studied a family with apparent autosomal dominant, early onset Hashimoto thyroiditis. Methods: The family was enrolled in an IRB-approved protocol. Whole exome sequencing was used to study the proband and an affected sibling. The identified variant was studied in other family members by Sanger sequencing. Results: We identified a previously unreported splice site variant in the thyroglobulin gene (TG c.10761G > C). This variant was confirmed in all affected family members who underwent testing, and also noted in one unaffected child. The variant is associated with exon 9 skipping, resulting in a novel inframe variant transcript of TG. Conclusion: We discovered a monogenic form of AITD associated with a splice site variant in the thyroglobulin gene. This finding raises questions about the origins of thyroid autoimmunity; possible explanations include increased immunogenicity of the mutated protein or thyroid toxicity with secondary development of anti-thyroid antibodies. Further study into the effects of this variant on thyroid function and thyroid autoimmunity are warranted. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction Autoimmune thyroid disease (AITD) includes both Graves disease and Hashimoto thyroiditis and is characterized by autoantibodies to thyroglobulin and thyroperoxidase. The etiology of AITD is complex and multifactorial [1]. Like many other autoimmune conditions, AITD disproportionately affects females and onset is in early adulthood, suggesting that hormonal and environmental factors are involved [2]. AITD is also known to be strongly heritable, and families with multiple affected siblings were first described in the 1960s [3]. Multiple genes have been identified through linkage analyses

* Corresponding author. Boston Children's Hospital, Fegan 7, 300 Longwood Ave, Boston, MA 02115, United States. E-mail address: [email protected] (M.S. Lo). https://doi.org/10.1016/j.jaut.2017.09.003 0896-8411/© 2017 Elsevier Ltd. All rights reserved.

and genome-wide association studies to contribute to AITD susceptibility. These include immune regulatory genes such as CTLA4, CD40, PTPN22, FOXP3, and CD25, which have also been implicated in the suspectibility toward other autoimmune diseases [4]. The HLADR3 allele also confers significant risk for AITD, perhaps by influencing presentation of thyroid self-antigen [5]. Variants in thyroid-specific genes, including TSHR (thyroidstimulating hormone receptor) and TG (thyroglobulin) are also associated with susceptibility to AITD [6,7]. Thyroglobulin, as the precursor for thyroid hormone, is the predominant protein within the thyroid gland [8]. Intronic and exonic TG polymorphisms have previously been linked to both Graves disease as well as Hashimoto thyroiditis phenotypes [9e11]. The TG SNPs previously described in association with AITD confer a relatively modest increase in risk; these gene variants are also found in healthy populations without AITD or anti-thyroid antibodies [10]. The mechanism by which these variants lead to autoimmunity is not known.

M.S. Lo et al. / Journal of Autoimmunity 86 (2018) 116e119

In this study, we used whole exome sequencing to evaluate an extended family with autosomal dominant inheritance of Hashimoto thyroiditis. We identified an extremely rare splice site variant in TG in affected members. 2. Methods 2.1. Subject recruitment The proband and affected and unaffected family members were enrolled in an IRB-approved study at Boston Children's Hospital. Informed consent was obtained from all enrolled subjects. 2.2. Exome sequencing Total genomic DNA was extracted from peripheral blood mononuclear cells using QIAmp DNA Mini Kit (Qiagen). DNA from the proband and one affected cousin was sent for whole exome sequencing (WES). WES was provided by the Yale University Centers for Mendelian Genomics on an Illumina HiSeq 2000. Libraries (TruSeq DNA v2 Sample Preparation kit; Illumina, San Diego, CA) and whole exome capture (EZ Exome 2.0, Roche) were performed according to manufacturer protocols. FASTQs were filtered, aligned, and variants were filtered and annotated by Codified Genomics (proprietary algorithm, Houston, TX). Sanger confirmation of the candidate variant was performed at the Boston Children's Hospital Gene Discovery Core. Likely pathogenic variants were selected to include nonsynonymous, splice site and indel variants with an allele frequency C) was identified. This is an extremely rare variant only seen once in the gnomAD database (mean allele frequency of 1/246,128, 0.0004%, accessed on July 12, 2017, gnomad.broadinstitute.org). Notably, no variants were identified in immune regulatory genes previously associated with autoimmune thyroiditis. The TG variant was confirmed by Sanger sequencing, also performed in additional family members (Fig. 1). All tested members with Hashimoto thyroiditis were found to carry the c.1076-1G > C variant. In contrast, all unaffected members lacked the mutation, with the exception of one unaffected sibling of the proband who also carried the variant. 3.3. mRNA expression of normal and c.1076-1G > C variant TG transcripts TG is a large gene, encompassing 48 exons with a cDNA product of 8.4 KB, resulting in an approximately 300 kDa protein (http:// www.ncbi.nlm.nih.gov/nuccore/NM_003235.4). The c.1076-1G > C variant was predicted to disrupt the splice site immediately preceding exon 9, which might cause in-frame deletion of the entire exon (Fig. 2A). To confirm the effect of this mutation, we examined TG transcripts in peripheral blood from a subject carrying the variant. Although TG is primarily expressed in thyroid tissue, the mRNA is also expressed in lymphocytes [12]. Therefore, we extracted RNA from peripheral blood lymphocytes isolated from the patient's mother, who also carries the c.1076-1G > C variant. PCR amplification of thyroglobulin cDNA using primers spanning exons 8 and 10 showed the presence of two cDNA isoforms in this heterozygous patient, one with and the other without exon 9 (Fig. 2B). The two isoforms were as predicted in size: 1.29 kb for the wild-type sequence, and 186 bp for the isoform lacking exon 9. Sanger sequencing of the 186 bp fragment further confirmed complete skipping of exon 9 (Fig. 2C). 4. Discussion To our knowledge, this is the first description of a family with true Mendelian inheritance of autoimmune thyroid disease (AITD). While familial AITD is a commonly recognized phenomenon, distinctive features of this family include the early age of onset, the lack of female predominance, and the fact that clinical phenotype was limited to Hashimoto thyroiditis and not Graves disease. Using a whole exome sequencing approach, we identified an autosomal dominant mutation in the thyroglobulin gene that results in an inframe deletion of exon 9. Functional characterization of this gene variant is still in progress, and we cannot yet state conclusively that the variant is causative of AITD. The thyroglobulin protein is thought to be the primary autoantigen in AITD, and single nucleotide polymorphisms (SNPs) in TG have been previously associated with thyroid autoimmunity [1]. It is possible that some TG SNPs influence thymic expression, leading to inadequate development of TG-specific regulatory T cells (Tregs). It has also been proposed that circulating TG levels, which could be influenced by genetic variation, may be important for maintenance of the TG-specific Treg population [13]. All of the TG SNPs previously associated with susceptibility for AITD are also found in healthy controls with relatively common frequency [7,9,14]. We are not aware of any prior reports of TG mutations leading directly to AITD in a Mendelian inheritance

118


Fig. 1. Pedigree of Hashimoto thyroiditis in a non-consanguineous family. Ages noted are current as of submission. Shading indicates diagnosis of Hashimoto thyroiditis. Arrow indicates proband. / indicates wild-type; þ/ indicates heterozygous for the c.1076-1G > C variant; NT indicates not tested.

Fig. 2. c.1076-1G > C variant results in in-frame deletion of exon 9. A, Exon structure of TG. Intronic regions are not depicted to scale. Arrowheads indicate primer locations in exons 8 and 10. B, PCR amplification of thyroglobulin cDNA from a healthy control (HC) and a c.1076-1G > C heterozygous subject, using primers shown in Fig. 2A. C, Sequencing chromatogram of 1.29 kb and 186 bp PCR products from c.1076-1G > C heterozygous patient.

pattern, although there are previous descriptions of families with autosomal dominant inheritance of anti-thyroid antibodies [15]. These reports described reduced disease penetrance in men, in contrast to the family presented here [15]. The c.1076-1G > C splice site variant in this family was only associated with Hashimoto thyroiditis and not Graves disease. It is unlikely that this is due merely to functional inactivity of the gene product, as studies of congenital hypothyroidism indicate that TG haploinsufficiency does not cause symptomatic disease [16,17]. One possible explanation for the consistent phenotype of thyroid insufficiency, as opposed to hyperactivity, in this family may be that

the mutated protein is dysfunctional but still able to form homodimers through the carboxyl-terminus acetylcholinesterase (ACHE)-like domain. It would thus interfere with dimerization of the normal protein as well, causing accumulation of TG within cells and consequent non-immune-mediated hypothyroidism. This mechanism would not clearly explain the development of antithyroid antibodies in the affected family members, however. Alternatively, the splice variant could result in a misfolded protein that is either toxic to the thyroid cell, causing increased cell turnover and eventually an immune response, or a protein that is directly immunogenic. Misfolding could uncover cryptic epitopes


that are aberrantly presented as self-antigen in the periphery, as hypothesized previously [18,19]. Finally, we note that although the correlation between genotype and phenotype appears strong in this family, there were two exceptions to the autosomal dominant inheritance pattern, suggestive of incomplete penetrance. There is precedent for incomplete penetrance in other monogenic autoimmune disorders. As an example, CTLA4 haploinsufficiency causes a syndrome of hypogammaglobulinemia with autoimmune features. The range and severity of organ system involvement is quite variable despite similar levels of CTLA-4 expression and function in vitro [20,21]. 5. Conclusion We describe here a family with early onset autoimmune thyroid disease associated with a splice site variant in the thyroglobulin gene. Functional studies are necessary to understand how this mutation leads to autoimmunity. Further exploration of mechanism may yield important insights in protein biochemistry and antigen presentation that may be applicable to other autoimmune conditions. Competing interests declaration The authors have no competing interests to declare. Acknowledgements We would like to thank the extended family for their participation in this case study and the authors gratefully acknowledge assistance and support from The Manton Center for Orphan Disease Research Gene Discovery Core. The work was made possible by National Institute of Health (NIH) grants R01 AR068429 (PBA), U19 HD077671 (PBA), and 5R01AI065617 (TAC). Sanger sequencing was performed by the Molecular Genetics Core Facility of the IDDRC at Boston Children's Hospital, supported by National Institutes of Health grant U54 HD090255 from NICHD. References [1] Y.H. Dong, D.G. Fu, Autoimmune thyroid disease: mechanism, genetics and current knowledge, Eur. Rev. Med. Pharmacol. Sci. 18 (2014) 3611e3618. [2] G. Effraimidis, W.M. Wiersinga, Mechanisms in endocrinology: autoimmune thyroid disease: old and new players, Eur. J. Endocrinol. 170 (2014) R241eR252.

119

[3] R. Hall, J.B. Stanbury, Familial studies of autoimmune thyroiditis, Clin. Exp. Immunol. 2 (1967). Suppl:719e725. [4] H.J. Lee, C.W. Li, S.S. Hammerstad, M. Stefan, Y. Tomer, Immunogenetics of autoimmune thyroid diseases: a comprehensive review, J. Autoimmun. 64 (2015) 82e90. [5] L. Muixi, M. Carrascal, I. Alvarez, X. Daura, M. Marti, M.P. Armengol, et al., Thyroglobulin peptides associate in vivo to HLA-DR in autoimmune thyroid glands, J. Immunol. 181 (2008) 795e807. [6] M. Stefan, C. Wei, A. Lombardi, C.W. Li, E.S. Concepcion, W.B. Inabnet 3rd, et al., Genetic-epigenetic dysregulation of thymic TSH receptor gene expression triggers thyroid autoimmunity, Proc. Natl. Acad. Sci. U. S. A. 111 (2014) 12562e12567. [7] Y. Tomer, D.A. Greenberg, E. Concepcion, Y. Ban, T.F. Davies, Thyroglobulin is a thyroid specific gene for the familial autoimmune thyroid diseases, J. Clin. Endocrinol. Metab. 87 (2002) 404e407. [8] B. Di Jeso, P. Arvan, Thyroglobulin from molecular and cellular biology to clinical endocrinology, Endocr. Rev. 37 (2016) 2e36. [9] Y. Ban, D.A. Greenberg, E. Concepcion, L. Skrabanek, R. Villanueva, Y. Tomer, Amino acid substitutions in the thyroglobulin gene are associated with susceptibility to human and murine autoimmune thyroid disease, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 15119e15124. [10] M. Caputo, C.M. Rivolta, T. Mories, J.J. Corrales, P. Galindo, R. Gonzalez-Sarmiento, et al., Analysis of thyroglobulin gene polymorphisms in patients with autoimmune thyroiditis, Endocrine 37 (2010) 389e395. [11] Y. Ban, T. Tozaki, M. Taniyama, L. Skrabanek, Y. Nakano, Y. Ban, et al., Multiple SNPs in intron 41 of thyroglobulin gene are associated with autoimmune thyroid disease in the Japanese population, PLoS One 7 (2012), e37501. [12] M. Amakawa, R. Kato, F. Kameko, M. Maruyama, J. Tajiri, Thyroglobulin mRNA expression in peripheral blood lymphocytes of healthy subjects and patients with thyroid disease, Clin. Chim. Acta 390 (2008) 97e103. [13] Y.M. Kong, N.K. Brown, G.P. Morris, J.C. Flynn, The essential role of circulating thyroglobulin in maintaining dominance of natural regulatory T cell function to prevent autoimmune thyroiditis, Horm. Metab. Res. 47 (2015) 711e720. [14] Y. Ban, T. Tozaki, M. Taniyama, M. Tomita, Y. Ban, Association of a thyroglobulin gene polymorphism with Hashimoto's thyroiditis in the Japanese population, Clin. Endocrinol. (Oxf.) 61 (2004) 263e268. [15] D. Phillips, S. McLachlan, A. Stephenson, D. Roberts, S. Moffitt, D. McDonald, et al., Autosomal dominant transmission of autoantibodies to thyroglobulin and thyroid peroxidase, J. Clin. Endocrinol. Metab. 70 (1990) 742e746. [16] C.E. Citterio, G.A. Machiavelli, M.B. Miras, L. Gruneiro-Papendieck, K. Lachlan, G. Sobrero, et al., New insights into thyroglobulin gene: molecular analysis of seven novel mutations associated with goiter and hypothyroidism, Mol. Cell. Endocrinol. 365 (2013) 277e291. [17] H.M. Targovnik, C.E. Citterio, C.M. Rivolta, Thyroglobulin gene mutations in congenital hypothyroidism, Horm. Res. Paediatr. 75 (2011) 311e321. [18] N. Arase, H. Arase, Cellular misfolded proteins rescued from degradation by MHC class II molecules are possible targets for autoimmune diseases, J. Biochem. 158 (2015) 367e372. [19] G. Carayanniotis, The cryptic self in thyroid autoimmunity: the paradigm of thyroglobulin, Autoimmunity 36 (2003) 423e428. [20] H.S. Kuehn, W. Ouyang, B. Lo, E.K. Deenick, J.E. Niemela, D.T. Avery, et al., Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4, Science 345 (2014) 1623e1627. [21] D. Schubert, C. Bode, R. Kenefeck, T.Z. Hou, J.B. Wing, A. Kennedy, et al., Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations, Nat. Med. 20 (2014) 1410e1416.