Genetic Variants Implicate Dual Oxidase-2 in Familial and Sporadic Nonmedullary Thyroid Cancer Darrin V

Published OnlineFirst September 9, 2019; DOI: 10.1158/0008-5472.CAN-19-0721

Cancer Genome and Epigenome Research

Genetic Variants Implicate Dual Oxidase-2 in Familial and Sporadic Nonmedullary Thyroid Cancer Darrin V. Bann1,2, Qunyan Jin3, Kathryn E. Sheldon2, Kenneth R. Houser4, Lan Nguyen2, Joshua I. Warrick5, Maria J. Baker6, James R. Broach2,4, Glenn S. Gerhard3,and David Goldenberg1

Abstract

Highly penetrant hereditary thyroid cancer manifests as rs965513[A] demonstrated higher DUOX2 expression than familial nonmedullary thyroid cancer (FNMTC), whereas heterozygous rs965513[A/G] or homozygous rs965513[A]- low-penetrance hereditary thyroid cancer manifests as negative patients. These data suggest that dysregulated sporadic disease and is associated with common poly- hydrogen peroxide metabolism is a common mechanism morphisms, including rs965513[A]. Whole-exome by which high- and low-penetrance genetic factors increase sequencing of an FNMTC kindred identiﬁed a novel thyroid cancer risk. Y1203H germline dual oxidase-2 (DUOX2) mutation. DUOX2Y1203H is enzymatically active, with increased Signiﬁcance: This study provides novel insights into the production of reactive oxygen species. Furthermore, genetic and molecular mechanisms underlying familial and patients with sporadic thyroid cancer homozygous for sporadic thyroid cancers.

Introduction cancer predisposition syndromes (9) and is thought to account for 3% to 10% of all thyroid cancers (10). FNMTC families display Thyroid cancer is the most common endocrine malignancy in a mix of benign and malignant thyroid tumors with an earlier age the United States, resulting in 62,450 new diagnoses and 1,950 of onset and a higher incidence of multifocal disease compared deaths in 2015 (1). Compared with other common cancers, with patients with sporadic thyroid cancer (10, 11). Germline thyroid cancer displays a high degree of heritability with first- mutations in HABP2, SRRM2, and FOXE1 have been reported in degree relatives having a 5.5-fold increase in relative risk of FNMTC kindreds (3–5); however, whether all of these mutations disease (2). This increased thyroid cancer risk results from both truly cause FNMTC remains controversial (12–14). high penetrance rare variants and low penetrance common In contrast to FNMTC, low-penetrance hereditary thyroid variants (3–8). Hereditary cancer syndromes associated with cancer has been associated with multiple common SNPs (6–8). medullary thyroid cancer have been well described; however, the The rs965513[A] allele, which confers the greatest relative genetic lesions associated with nonmedullary thyroid cancer riskforthedevelopmentofthyroid cancer (6, 7), resides within heritability are less well defined. Familial nonmedullary thyroid an enhancer element controlling expression of FOXE1 (15, 16), cancer (FNMTC) is defined as thyroid cancer of follicular cell athyroid-specific transcription factor that regulates several origin in two or more first-degree relatives in the absence of other genes including thyroid peroxidase (TPO), thyroglobulin (TG), the sodium-iodide symporter (SLC5A5), and dual oxi- 1 — Department of Otolaryngology Head & Neck Surgery, The Pennsylvania dase-2 (DUOX2; ref. 17). The risk allele is associated with State University, College of Medicine, Hershey, Pennsylvania. 2Institute for Personalized Medicine, The Pennsylvania State University, College of Medicine, decreased expression of FOXE1 and siRNA knockdown of Hershey, Pennsylvania. 3Lewis Katz School of Medicine at Temple University, FOXE1 causes altered expression of DUOX2, TPO, TG, and Philadelphia, Pennsylvania. 4Department of Biochemistry and Molecular SLC5A5 in vitro (17). Biology, The Pennsylvania State University, College of Medicine, Hershey, Using whole-exome sequencing, we identified a novel Pennsylvania. 5Department of Pathology, The Pennsylvania State University, 6 Y1203H germline DUOX2 mutation in an FNMTC kindred. College of Medicine, Hershey, Pennsylvania. Department of Medicine, Division DUOX2 generates hydrogen peroxide (H O ), which is used by of Hematology/Oncology, The Pennsylvania State University, College of Med- 2 2 icine, Hershey, Pennsylvania. TPO to iodinate tyrosyl residues on TG for thyroxine (T4) and 3,30,5-triiodothyronine (T3)synthesis,and loss-of-function muta- Note: Supplementary data for this article are available at Cancer Research tions in DUOX2 cause congenital hypothyroidism (18). We Online (http://cancerres.aacrjournals.org/). report that DUOX2Y1203H is functionally active and is highly Corresponding Author: David Goldenberg, The Pennsylvania State University, expressed in normal thyroid tissue, potentially contributing to College of Medicine, 500 University Drive, H091, Hershey, PA 17033. Phone: 717- 531-8946; Fax: 717-531-6160; E-mail: [email protected] increased tumorigenesis. Strikingly, DUOX2 expression is increased in patients with rs965513[A] homozygosity, consistent Cancer Res 2019;79:5490–9 with a model in which DUOX2-mediated dysregulation of H2O2 doi: 10.1158/0008-5472.CAN-19-0721 metabolism underlies both high- and low-penetrance hereditary 2019 American Association for Cancer Research. thyroid cancers.

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DUOX2 Variant Causes Hereditary Thyroid Cancer

Materials and Methods expressed in thyroid tissue across all three data sets. Differential expression was defined by E > 1:5ðQ Q Þþ Q3 where E Subjects were recruited at the Penn State College of Medicine. i 3 1 i represents expression of gene i in thyroid tissue, Q represents The study was approved by the Penn State College of Medicine 3 the third quartile expression value for gene i across all tissues, and Institutional Review Board. Written informed consent was Q represents the first quartile gene expression value for gene i obtained from all participants whose DNA and tumor tissues 1 across all tissues. Variants occurring in genes where the raw were analyzed. expression value was less than the mean Q1 value for all genes in thyroid tissue across all three databases were also excluded. Nucleic acid extraction, whole-exome capture, and next- PolyPhen-2 (28) scores were used to identify variants with poten- generation sequencing tial functional impact, and variants with a score <0.3 were Germline DNA was extracted from whole blood or saliva using excluded. For the remaining variants, literature searches were a Qiagen QIAsymphony nucleic acid isolation robot. Qiagen conducted to identify any potential function for impacted genes AllPrep DNA/RNA Mini Kits were used to extract nucleic acids in the thyroid gland; only genes directly implicated in thyroid from tumor and normal tissue stored in RNA later at 80 C gland physiology were included. Further testing required that (Thermo Fisher Scientific) or liquid nitrogen. Additional details candidate SNPs be absent in an unaffected and unrelated spouse are provided in Supplementary Methods. and present in an additional affected family member. Genomic DNA was sheared into approximately 200-bp frag- ments and processed into Illumina-compatible sequencing librar- Somatic mutation detection ies with a PrepX ILM DNA Library Kit (WaferGen) on an Apollo Somatic mutations in tumor exomes were detected using 324 instrument (WaferGen). Sheared DNA (500–1,000 ng) was MuTect v1.1.7 (29). To reduce the incidence of false-positive processed into Illumina-compatible sequencing libraries with a mutation calls due to sequencing artifacts, alignment errors, and PrepX ILM DNA Library Kit (WaferGen) on an Apollo 324 rare germline mutations, the "panel of normals" flag was imple- instrument (WaferGen). Libraries were uniquely bar coded, mented as described (30) using white blood cell whole-exome amplified, and subjected to multiplex SeqCap EZ Exome V3.0 sequence data from 50 patients with no known hematologic (Roche) hybridization and capture. Libraries were sequenced on malignancy. Details regarding the "panel of normals" can be an Illumina 2500 (Illumina Inc.) using 2 100 bp reads in high- found in Supplementary Methods. Filtered Variant Call Format output mode. Sequence data have been deposited at the European files were annotated using TabAnno v2.13 (31) and somatic Genome–phenome Archive (EGA), which is hosted by the Euro- mutations were confirmed by visual inspection in Integrative pean Bioinformatics Institute (EBI) and the Centre for Genomic Genomics Viewer (32). Regulation, under accession number EGAS00001003782. Further information about EGA can be found on https://ega-archive.org. Protein modeling and alignment "The European Genome–phenome Archive of human data con- Modeling of the DUOX2 peroxidase domain (NCBI accession sented for biomedical research" (http://www.nature.com/ng/jour NP_054799.4) was performed using SWISS-MODEL (33). nal/v47/n7/full/ng.3312.html). Additional details about library Amino acid sequences of DUOX2 or the orthologous preparation and sequencing are provided in Supplementary protein were obtained for the following species: Homo sapiens Methods. (NCBI accession NP_054799.4), Pan troglodytes (NCBI accession XP_009427327.1), Macaca mulatta (NCBI accession Sequencing data pipeline and variant filtering XP_014997623.1), Canis lupus familiaris (NCBI accession XP_ Next-generation sequencing data were processed according to 013964824.1), Bos taurus (NCBI accession XP_010807708.1), Genome Analysis Toolkit (GATK) best practices recommenda- Mus musculus (NCBI accession NP_808278.2), Rattus norvegicus tions using GATK v2.7.4 (19–21). Variants within the (NCBI accession NP_077055.1), Drosophila melanogaster (NCBI exome capture region 100 bp were identified using the GATK accession NP_001259968.1), and Caenorhabditis elegans (NCBI Haplotype Caller module v3.3.0 (20, 21). Additional details are accession NP_490684.1). Protein alignment was performed using provided in Supplementary Methods. Germline variants were Clustal Omega (34). filtered using Golden Helix SNP and Variation Suite v8.1.5 (Golden Helix, Inc.) according to the following criteria: (i) seg- PCR, Sanger sequencing, and targeted deep sequencing regation with an autosomal dominant mechanism of inheritance; Sanger sequencing was used to verify variants identified by (ii) SNPs produced a nonsynonymous variation in a defined next-generation exome sequencing. PCR amplification of regions < exon; (iii) SNPs were present in 0.5% of the population encom- spanning DUOX2Y1203, rs965513, BRAFV600, KRASQ61, HRASQ61, passed by the 1,000 Genomes Project Phase 1, the dbSNP build and NRASQ61 was conducted using primers shown in Supple- 137 common variants database (http://www.ncbi.nlm.nih.gov/ mentary Table S1. Details regarding PCR conditions are described SNP; ref. 22), the NHLBI GO Exome Sequencing Project build 138 in Supplemental Methods. Sanger sequencing was conducted database (23), and the Exome Aggregation Consortium database by GENEWIZ LLC. Mutation status was determined by visual version 0.3.1 (24); and (iv) SNPs had a variant quality score >20 inspection of fluorescence trace files of capillary electrophoresis. > and read depth 10. Targeted deep sequencing libraries of gel-purified BRAFV600 and fi We next ltered for variants in genes that had a previously KRASQ61 PCR products were constructed using the KAPA hyper known function reported in PubMed and for those highly prep kit (KAPA Biosystems). Libraries were sequenced on an expressed in thyroid tissue. Tissue-specific gene expression data Illumina MiSeq instrument generating 2 250 bp reads. Adapter were derived from Illumina Body Map 2.0, GTEx, and Uhlen's lab sequences were trimmed and reads were aligned to the hg19 experiments in the EMBL-EBI Expression Atlas Database (25–27). reference genome using the FASTQ Toolkit and BWA Aligner apps Variants were retained if they occurred within a gene differentially on the Illumina BaseSpace website (http://basespace.illumina.

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com). Minor allele frequencies were determined by manual Devices). All experiments were performed in triplicate. ROS inspection of BAM files in Integrative Genomics Viewer (32). production for each transfection condition was standardized to the empty vector transfection negative control. Statistical differ- Quantitative reverse transcription PCR ences in ROS production were calculated using Students t test as Extracted RNA was DNAse treated using an Ambion TURBO implemented in Microsoft Excel (Microsoft Corporation). DNA-free kit (Thermo Fisher Scientific) and reverse transcribed Similar protein expression across experiments was confirmed using an Applied Biosystems High-Capacity cDNA Reverse using semiquantitative Western blotting of cells transfected in Transcription Kit (Thermo Fisher Scientific) according to the parallel. Briefly, cell extracts were prepared in RIPA buffer (Sigma) manufacturer's instructions. Quantitative PCR was performed supplemented with protease inhibitor cocktail (Sigma), and using Applied Biosystems (Thermo Fisher Scientific) TaqMan protein concentrations were determined by the BCA method. Gene Expression Master Mix and TaqMan Probes targeting Equal quantities of protein input (30 mg) were separated by FOXE1 (catalog no. Hs00916085-S1), DUOX2 (catalog no. 4% to 12% Nupage Tris-Bis gel electrophoresis with SeeBlue Plus Hs00204187-m1), TPO (catalog no. Hs0892518-m1), TG 2 Protein Standard (Invitrogen) under reducing conditions and (catalog no. Hs00794359-m1), and GAPDH (catalog no. transferred to an Immobilon-P transfer membrane (EMD Hs02758991-G1). Reactions were analyzed on an Applied Millipore). Immunoblots were incubated with DUOX2 S-12 Biosystems QuantStudio 12K Flex Instrument (Thermo Fisher (MABN787; EMD Millipore) diluted 1:1,000, GAPDH diluted Scientific). Expression of each target was determined as fold 1:1,000 (sc-98501; Santa Cruz Biotechnology), and DUOXA2 expression relative to GAPDH averaged over three independent diluted 1:100 (sc-98501; Santa Cruz Biotechnology). Blots were replicates. Differences in gene expression relative to rs965513 processed using a ScanLater kit with appropriate secondary anti- genotype were computed using the Kruskal–Wallis rank sum test bodies according to the manufacturer's instructions (Molecular as implemented by the "stats" package in R v3.2.2 (35). Devices, LLC) and quantified using ImageJ (36).

Plasmids and mutagenesis IHC The vectors EX-NEG-M02, human DUOX2 (EX-E1601-M02), Slides were prepared from 5-mm sections of formalin-fixed and human DUOXA2 (EX-H0633-M02) were purchased from paraffin-embedded tissue. Representative sections were stained Genecopoeia. The DUOX2Y1203H expression vector was prepared with hematoxylin and eosin and fitted with glass coverslips for from the wild-type DUOX2 cDNA using the QuikChange Light- imaging. Remaining samples were deparaffinized using standard ning Site-Directed Mutagenesis Kit (Agilent) with forward primer procedures and antigen retrieval was performed by incubation in 50-gggaggcgaagacatgcatgatggccaggac-30 and reverse primer 50- 10 mmol/L sodium citrate, pH 6.0 with 1 mmol/L EDTA at 95C gtcctggccatcatgcatgtcttcgcctccc-30 according to the manufacturer's for 20 minutes. Samples were incubated with mouse anti-DUOX2 protocols. Following mutagenesis, the construct was confirmed S-12 monoclonal antibody (0.5 mg/mL, diluted 1:1,000; by bidirectional Sanger sequencing. MABN787; EMD Millipore) at 4C overnight. Slides were then incubated with undiluted ImmPRESS goat antimouse-HRP sec- Cell culture and stable transfections ondary antibody (Vector Laboratories) for 30 minutes at room Cos7 (CRL1651, ATCC) cells were cultured in DMEM (ATCC) temperature, washed, and developed with diaminobenzidine with 10% fetal bovine serum (ATCC) under 5% CO2/95% air at (DAB; Vector Laboratories). Slides were counterstained with 37C. To produce stable cell lines, Cos7 cells were transfected with hematoxylin prior to being fitted with glass coverslips for imag- wild-type human DUOXA2 in a cytomegalovirus-driven expres- ing. Slides were reviewed by a board-certified head and neck sion vector (pReceiver-M02) or empty vector using Lipofectamine pathologist (Joshua I. Warrick). 3000 (Thermo Fisher Scientific) transfection reagent according to the manufacturer's protocol. Forty-eight hours after transfection, cells were replated in medium with Geneticin (G418; Thermo Results Fisher Scientific; 500 mg/mL) for selection. After 2 weeks of Identification of a novel missense mutation associated with selection, cells were pooled, expanded, and maintained with FNMTC Geneticin (G418; 250 mg/mL). The proband (Fig. 1A, patient III.2) presented with multifocal micropapillary thyroid carcinoma at 46 years of age. She had an Reactive oxygen species measurements extensive family history of thyroid cancer. The proband's mater- The multiplex ROS-Glo H2O2 Assay (Promega) and the Amplex nal grandmother (Fig. 1A, patient I.2), who ultimately succumbed Red Hydrogen Peroxide/Peroxidase Assay (Thermo Fisher Scien- to the disease, had three children (II.2, II.3, and II.4) all of whom tific) were used to measure extracellular H2O2,, whereas intracel- were diagnosed with thyroid cancer. Individual II.2 has been lular reactive oxygen species (ROS) were measured using a treated for highly aggressive, recurrent papillary thyroid cancer DCFDA ROS Detection Assay kit (AbCam). For all assays, Cos7 (PTC) at our institution (Fig. 1A). Individuals II.1 and II.2 had cells stably expressing DuoxA2 were plated in 96-well plates and four children, all of whom were affected: two with papillary grown to 70% to 80% confluence according to the manufacturer's thyroid cancer (proband and III.4), and two with nodular thyroid protocol. Cells were then transiently transfected with the EX-NEG- disease (III.3 and III.5; Fig. 1A). The disease status of individuals M02 empty vector, DUOX2 vector, or the DUOX2Y1203H vector III.6, III.7, and III.8 is not known. The proband (III.2) has two using Lipofectamine 3000 (Thermo Fisher Scientific) according to children (IV.1 and IV.2), one of whom (IV.1) was diagnosed with the manufacture's protocol. Twenty-four hours after transfection, a benign thyroid nodule by ultrasound examination at age the ROS and cell viability assays were performed according to the 19 years (Fig. 1A). Importantly, no patient in this pedigree manufacturer's instructions. Relative luminescence was deter- exhibited clinical symptoms of hypothyroidism. The prethyroi- mined using a SpectraMax I3 Multiplate reader (Molecular dectomy thyroid-stimulating hormone (TSH) level for the

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DUOX2 Variant Causes Hereditary Thyroid Cancer

Figure 1. Familial nonmedullary thyroid cancer associated with a novel mutation in DUOX2. A, Pedigree of a kindred with highly penetrant autosomal-dominant FNMTC across four generations. The key on the lower right indicates disease status for each patient. The proband patient is indicated by the arrow and the age of each patient at diagnosis is shown to the right. The allele status of Chr15:15:45,389,898 is shown below selected patients and was conﬁrmed by examining electropherograms from Sanger sequencing. Patients subjected to whole-exome sequencing are indicated by the dashed lines. B, A cartoon depiction of DUOX2 and its cognate maturation factor DUOXA2. The star indicates the approximate location of the Y1203H mutation. C, Amino acid sequence alignments from the DUOX2 protein of nine vertebrate and invertebrate species demonstrate that Y1203 is highly conserved among mammals. Amino acid positions from human DUOX2 are shown below the alignment and the red box indicates Y1203. Transmembrane domains are shown as blue shading. D, Cos7 cells were stably transfected with DUOXA2 (Cos7-DUOXA2) or empty vector (Cos7-EV) and transiently transfected with empty vector (EV), wild-type (WT) DUOX2, or

DUOX2Y1203H. ROS production was measured as a function of cell viability using three assays: RosGlo (extracellular ROS), Amplex Red (extracellular ROS), and DCFDA (intracellular ROS). The graphs show the mean relative ﬂuorescence value of three independent experiments, with error bars indicating SEM. Data were normalized to the baseline established by EV transfection, which was set to a value of 1. , P < 0.05. E, A representative Western blot from the experiment

outlined in D showing similar expression of wild-type DUOX2 and DUOX2Y1203H in Cos7-DUOXA2 cells. Transient transfection of wild-type DUOX2 or DUOX2Y1203H into Cos7-EV cells resulted in minimal expression of DUOX2 or DUOX2Y1203H. Expression of wild-type or mutant DUOX2 relative to GAPDH is shown below each lane.

proband patient was 4.4 mIU/mL (reference range: 0.35– cause a Y1203H substitution in DUOX2 (Table 1), was iden- 5.5 mIU/mL), although preoperative TSH levels were not available tified in all affected individuals (Fig. 1A). Sanger sequencing for the other individuals in the study. This pedigree is consistent confirmed the presence of the variant in all affected individuals with autosomal dominant hereditary papillary thyroid cancer but not in an unaffected family member (Fig. 1A, patient III.1). (Fig. 1A). The probability of this mutation occurring in all affected family Germline DNA from five affected individuals (Fig. 1A, members is approximately 1.6%. Analysis of the dbSNP data- dashed lines) was subjected to whole-exome sequencing. After base (22) revealed that the prevalence of this mutation in quality, frequency, segregation, and functional filtering, mis- the general population is approximately 1/138,000 indivi- sense variants were identified in three genes: DUOX2, IFT140, duals, indicating that DUOX2Y1203H is an extremely rare muta- and WDR72 (Table 1). Because of its central role in thyroid tion. No hereditary cancer predisposition syndromes known to physiology, we focused our analysis on DUOX2. A single, cause thyroid cancers were detected in affected individuals heterozygous A to G variant at chr15:45,389,898,predictedto (Supplementary Table S2). Furthermore, all sequenced

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Table 1. Whole-exome sequencing variant ﬁltering protocol Filtering step Mean coverage depth Number of variants 1. Variants identiﬁed * Patient II.2 107.3 644,267 * Patient III.2 68.1 541,180 * Patient III.3 90.4 608,064 * Patient III.4 112.7 1,728,519 * Patient III.5 94.4 1,826,547 2. Heterozygous variants present in all affected family members 33,234 3. Nonsynonymous variants in protein coding exons 1,560 4. Variants in <0.5% of population represented by Exome Variant Server, dbSNP, and 1,000 genomes 838 databases 5. Genotype quality >20 and read depth >20 823 6. Variant in a highly expressed gene that is differentially expressed in thyroid tissue 6 7. PolyPhen-2 score > 0.300 3

Gene Location Nucleotide Amino acid Notes DUOX2 Chr15:45,389,898 c.3607T>C p.Y1203H H2O2-generating NADPH oxidase, highly expressed in thyrocytes IFT140 Chr16:1,574,650 c.3044A>C p.H1015P Flagellar transport gene. Mutation results in retinal dystrophy or cystic kidney disease. WDR72 Chr15:53,994,442 c.1458C>A p.D486E Mutations associated with amelogenesis imperfecta include Val491Dfs, S783Ter, W978Ter, and S953Vfs. No evidence for speciﬁc function in thyroid, may be involved in parathyroid function

individuals were negative for the HABP2G534E,FOXE1A248G, Moreover, wild-type and mutant DUOX2 were expressed at low and SRRM2S346E polymorphisms previously implicated in levels in Cos7 cells stably transfected with empty vector FNMTC (Supplementary Table S2; refs. 3, 4). (Fig. 1E, Cos7-EV), confirming that DUOX2Y1203H expression The DUOX2 protein consists of an extracellular N-terminal is dependent upon DUOXA2 as expected (37). These data peroxidase domain; seven transmembrane domains containing demonstrate that DUOX2Y1203H contains a functional peroxi- four invariant histidines, which may be coordination sites for two dase domain capable of producing H2O2 and suggest that nonidentical prosthetic heme moieties (37); two EF-Hand calci- DUOX2Y1203H increases extracellular ROS compared with um-binding domains; and intracellular C-terminal FAD- and wild-type DUOX2. NADPH-binding domains (Fig. 1B). Expression, proper localiza- tion, and function of DUOX2 are dependent on its cognate DUOX2 expression in normal thyroid and thyroid tumor tissue maturation factor, DUOXA2 (Fig. 1B), which interacts with the We next sought to determine the effect of DUOX2Y1203H on extracellular "A" loop of DUOX2 (37). The Y1203H mutation is normal thyroid and thyroid tumor histology by comparing located within the fifth transmembrane domain, which is distant tissue samples from the proband patient (Fig. 1A, patient III.2) from the enzymatic active site and the DUOX2A interaction and a patient with sporadic thyroid cancer. Upon hematoxylin domain (Fig. 1B), but is highly conserved across mammalian and eosin staining, nontumor tissue from the proband patient species (Fig. 1C). was normal in appearance (Fig. 2A) and was similar to nontumor thyroid tissue from the patient with sporadic thyroid Functional analysis of DUOX2Y1203H cancer (Fig. 2B). Histologic examination of the proband's Previous reports have described DUOX2 mutations, predom- thyroid gland revealed two distinct tumors (Fig. 2A): a 7-mm inantly in the extracellular peroxidase domain or the intracel- tumor with follicular variant papillary thyroid cancer architec- lular calcium-binding domain, resulting in loss of function and ture (Fig. 2A, Micro-PTC 1), and a 4-mm tumor with classical congenital hypothyroidism (37). We therefore tested whether papillary architecture (PTC; Fig. 2A, Micro-PTC 2). A single the DUOX2Y1203H variant disrupts the H2O2 and ROS- tumor with classical papillary architecture was identified in the generating capacity of the enzyme. Because DUOX2 expression thyroid gland extirpated from the patient with sporadic thyroid and H2O2 synthesis requires coexpression of DUOXA2 (37), we cancer (Fig. 2B). generated a Cos7 cell line that stably expressed human DUOX2 is highly expressed in thyroid follicular cells (25, 38). DUOXA2 (Cos7-DUOXA2). After transfection of Cos7- To determine the effect of the Y1203H substitution on DUOX2 DUOXA2 cells with empty vector, wild-type DUOX2, or expression, we performed IHC to compare DUOX2 expression DUOX2Y1203H, extracellular H2O2 production was measured between the proband and the patient with sporadic thyroid using independent RosGlo and Amplex Red assays, whereas cancer. Antibody specificity was confirmed by Western blotting intracellular global ROS levels were measured using the (Fig. 1E). Thyroid follicular cells stained strongly for DUOX2 in DCFDA ROS detection assay (Fig. 1D). Wild-type DUOX2 and normal tissue from the proband (Fig. 2A, a-DUOX2) and a DUOX2Y1203H significantly increased extracellular and intracel- patient with unrelated sporadic thyroid cancer with wild-type lular H2O2 and ROS levels relative to empty vector controls. DUOX2 (Fig. 2B, a-DUOX2). The proband's MicroPTC-1 lesion The Amplex Red assay demonstrated increased extracellular exhibited strong DUOX2 staining, although expression was gross- H2O2 production by DUOX2Y1203H compared with wild-type ly reduced in MicroPTC-2 (Fig. 2A), suggestive of clonally distinct DUOX2. Western blotting confirmed similar expression of tumors arising within a single individual. The tumor isolated from wild-type and mutant DUOX2 in each experiment (Fig. 1E). the patient with sporadic PTC exhibited DUOX2 expression

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Figure 2. DUOX2 expression and mutational proﬁle of normal and thyroid tumor tissue in patients with familial or sporadic thyroid cancer. A, Photomicrographs depicting normal thyroid and two thyroid tumors from the proband patient. Slides were stained with hematoxylin and eosin (H&E) or were subjected to IHC with a-DUOX2

antibodies (a-DUOX2). Targeted next-generation sequencing to a depth of >15,000 was used to demonstrate exclusivity of the KRASQ61R mutation in Micro- PTC1 and the BRAFV600E mutation in Micro-PTC2. Below each photomicrograph, raw read counts are shown for wild-type (WT count, black) and mutant (Mut. count, red) KRASQ61 and BRAFV600. Graphs depict the percentage of reads corresponding to wild-type (black) or mutant (red) KRASQ61 and BRAFV600. B, Photomicrographs of normal thyroid and thyroid tumor tissue from a patient with wild-type DUOX2 who developed thyroid cancer with a BRAFV600E oncogenic driver mutation. Slides were stained with hematoxylin and eosin or were subjected to IHC with a-DUOX2 antibodies. The exclusivity of this mutation within tumor tissue was conﬁrmed by targeted next-generation sequencing to a depth of >600. Raw read counts corresponding to wild-type (WT count, black)

and mutant (Mut. count, red) KRASQ61 and BRAFV600 are shown below each photomicrograph. Graphs depict the percentage of reads corresponding to wild- type (black) or mutant (red) KRASQ61 and BRAFV600.

similar to adjacent normal thyroid tissue (Fig. 2B). Together, these consequently DUOX2, in patients with the rs965513[A] poly- data indicate that DUOX2Y1203H heterozygosity does not dramat- morphism may underlie sporadic thyroid cancer susceptibility. ically reduce DUOX2 expression, although expression of DUOX2 To test this hypothesis, we genotyped 64 patients with thyroid may vary depending upon the differentiation pattern of thyroid cancer for the rs965513 allele using Sanger sequencing and tumors. quantified expression of FOXE1, DUOX2, and other thyroid- To determine whether DUOX2Y1203H was associated with DNA specific genes in normal thyroid tissue by quantitative PCR. mutagenesis in the thyroid gland, we profiled the mutational The A/G genotype was the most common (n ¼ 32, 50%), spectrum of tumor tissue and adjacent normal tissue from the followed by the G/G genotype (n ¼ 17, 27%) and the A/A proband and the patient with sporadic thyroid cancer using genotype (n ¼ 15, 23%). Expression of FOXE1 was lower in the whole-exome sequencing. The MicroPTC-1 tumor from the pro- high-risk A/A group than in the G/G and A/G genotypes, band was positive for the KRASQ61R oncogenic driver mutation although this difference did not reach statistical significance whereas the MicroPTC-2 tumor was positive for the BRAFV600E (Fig.3A).HigherexpressionofDUOX2(P ¼ 0.033), TPO driver mutation (Fig. 2A). The mutual exclusivity of oncogenic (P ¼ 0.031), TG (P ¼ 0.029), and SLC5A5 (P ¼ 0.014) was driver mutations in the two tumors was confirmed by Sanger found in the A/A group (Fig. 3B–E), consistent with decreased sequencing and targeted next-generation sequencing of the FOXE1 promoter activity (8). mutated regions to a depth of >15,000 (Fig. 2A). Sequence analysis of the sporadic tumor revealed a single BRAF muta- V600E Discussion tion (Fig. 2B). The exclusivity BRAFV600E oncogenic driver mutation was confirmed by targeted next-generation sequencing Few genes have been identified as bona fide causes of FNMTC, (Fig. 2B). partially due to the low prevalence of familial disease. The estimated prevalence of thyroid cancer in the United States is The rs965513[A] thyroid cancer risk polymorphism is 0.14% (1), with FNMTC accounting for 3% to 10% of cases (10). associated with increased DUOX2 expression Thus, only 0.0042% to 0.014% of individuals in the general The mechanism through which the rs965513[A] polymor- population are likely to be affected by FNMTC in their lifetime. phism influences risk of nonmedullary thyroid cancer is not Our analysis of a single highly penetrant pedigree using exome known. We hypothesized that altered expression of FOXE1, and sequencing identified a novel germline Y1203H missense variant

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in DUOX2 that segregated as an autosomal dominant thyroid cancer phenotype. The DUOX2Y1203H variant seems to be a very rare mutation that has been identiﬁed in only 1 of 138,000 individuals in the dbSNP database (22), consistent with its role as a causative mutation in a rare condition such as FNMTC. Other germline variants, including HABP2G534E, SRRM2S346F, and FOXE1A248G (3–5), have been reported to cause FNMTC. The prevalence of HABP2G534E SNP at 1% to 5% (13, 14, 22, 39) and FOXE1A248G at 5.3 of 1000 individuals (22) is not consistent with the predicted prevalence of FNMTC at 1 of approximately 10,000 individuals in the general population (10). The SRRM2S346F polymorphism is more rare with a predicted prevalence of 6 of 10,000 individuals (22), however, the mechanism by which this mutation induces thyroid cancer remains unknown (5). Given that FNMTC is an uncommon disease, true causative mutations may be poorly represented, even in large datasets, consistent with the apparent rarity of DUOX2Y1203H (22). Fur- thermore, the observation that all cases of FNMTC reported thus far are associated with distinct germline mutations suggests that the FNMTC phenotype may be the common endpoint resulting from a variety of genetic lesions. A potential role for DUOX2 in thyroid cancer has recently been suggested (40). The DUOX2Y1203H variant is a compelling potential cause of FNMTC because DUOX2 is the major enzyme that generates H2O2 required for thyroid hormone synthesis in the thyroid gland. Hydrogen peroxide may be involved in carcino- genesis via multiple mechanisms including oxidative DNA damage, cell signaling, cell proliferation, regulation of gene expression, and cell senescence and apoptosis (40). Interestingly, H2O2 directly damages both DNA and RNA, leading to DNA and RNA mutagenesis and increased turnover (41). Increased DNA mutagenesis in association with DUOX2Y1203H is supported by the ﬁnding that the two thyroid tumors in the proband are genetically independent and have distinct oncogenic driver mutations (Fig. 2A). Multifocality is thought to be more common among patients with FNMTC than among patients with sporadic thyroid cancer (10, 11). Tumorigenesis is a complex process and should therefore be a rare event (42, 43). The presence of multiple genetically independent tumors within a single thyroid gland raises the intriguing possibility that patients with multifocal thyroid cancer, particularly those with a family history of thyroid cancer, may have a genetic predisposition toward thyroid tumorigenesis. The precise mechanism underlying DUOX2Y1203H-mediated tumorigenesis remains to be determined; however, in vitro experiments suggest that Y1203H increases H2O2 production (Fig. 1D). Interestingly, two assays that measure extracellular ROS (ROSGlo and Amplex Red) indicate that DUOX2Y1203H increases extracellular ROS levels compared with wild-type DUOX2, whereas measurement of

Figure 3. Effect of rs965513 genotype on thyroid-speciﬁc gene expression. Sanger sequencing was used to determine the rs965513 genotype for 64 patients with papillary thyroid cancer. Messenger RNA expression levels were determined for FOXE1 (A), DUOX2 (B), TPO (C), TG (D), and SLC5A5 (E)and are expressed as log-fold expression relative to GAPDH controls. Each point indicates gene expression from an individual patient, determined as the mean of three independent experiments. Patients were divided by rs965513 genotype as shown below each graph. Box plots indicate median expression values, with hinges at the upper and lower quartile and whiskers extending to 1.5 the interquartile range. , P < 0.05.

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intracellular ROS using DCFDA demonstrated similar ROS pro- were homozygous for rs965513[A], more than double the duction between wild-type and mutant DUOX2. These data expected 11% to 12% prevalence of rs965513[A] homozygosity suggest that the DUOX2Y1203H mutation may result in dysregu- in the general population (6, 22). We found that individuals lated ROS production at the plasma membrane; however, further homozygous for rs965513[A] expressed higher levels of DUOX2, studies are required to evaluate this possibility. Although the TPO, TG, and SLC5A5 (Fig. 3) than heterozygous rs965513[A/G] differences in ROS production that we observed were small and or homozygous-reference rs965513[G] individuals. These data measured over a short time period (24 hours), changes in ROS are consistent with a potential mechanism for the rs965513[A] metabolism that persist over many years may have cumulative allele to increase thyroid cancer susceptibility via enhanced biological effects that contribute to thyroid tumorigenesis, which DUOX2 expression and H2O2 production. Similar mechanisms occurs over the course of decades. may therefore underlie highly penetrant FNMTC and some cases Homozygous or compound heterozygous mutations that of PTC caused by low-penetrance risk factors including compromise DUOX2 enzymatic activity and result in congen- rs965513[A]. ital hypothyroidism are found primarily in the N-terminal We note the significant intragroup variability in gene expres- extracellular peroxidase-like domain and in the EF hand sion. Given that each data point represents a mean of three domains (18, 38, 44). By contrast, the tyrosine at position independent experiments, it is likely that there is true biological 1203 resides in the fifth transmembrane domain. To date, variability in gene expression among the population, although only three DUOX2 transmembrane mutations have been iden- the underlying reasons are not known. Possibilities for variability tified in association with congenital hypothyroidism, all in the in thyroid-specific gene expression may include undiagnosed context of compound heterozygosity for other damaging thyroid pathology, such as hypothyroidism or autoimmune thy- mutations (45–47). There was no evidence of clinical hypo- roid disease, and environmental effects. thyroidism in the kindred we analyzed. In summary, we used an unbiased approach to identify a The transmembrane location of the Y1203H mutation raises novel germline mutation in DUOX2 associated with highly the possibility that DUOX2Y1203H is mislocalized, resulting penetrant FNMTC. Combined with evidence that the FOXE1 in dysregulated H2O2 metabolism. Indeed, other mutations in rs965513[A] polymorphism also increases DUOX2 expression, DUOX2 have been described that result in mislocalization of our data suggest that dysregulation of proteins involved in an enzymatically active protein to an intracellular compart- H2O2 metabolism may be a common mechanism underlying ment and presumed enhanced degradation (44, 48). However, common high- and rare low-penetrance genetic factors that 2þ it is also possible that because H2O2 generation is Ca -depen- increase thyroid cancer susceptibility. This observation has dent (49), enzyme function may be enhanced by increased translational implications for screening, chemoprevention, and calcium binding or availability in different subcellular loca- development of therapeutics. tions. Increased oxidative damage from H2O2 produced by DUOX2Y1203H in vivo may also result from disruption of Disclosure of Potential Conflicts of Interest the physiologic colocalization of DUOX2 and TPO at the apical No potential conflicts of interest were disclosed. plasma membrane, although free H2O2 can lead to DUOX2 inactivation (50). It is notable that the Y1203H mutation Authors' Contributions is near two transmembrane heme moieties that coordinate Conception and design: D.V. Bann, J.R. Broach, G.S. Gerhard, D. Goldenberg via histidine residues (37). The Y1203H substitution may Development of methodology: D.V. Bann, Q. Jin, K.E. Sheldon, L. Nguyen, therefore impact heme binding, although the effect of altered D. Goldenberg heme binding on protein function is not known. Future studies Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D.V. Bann, Q. Jin, K.R. Houser, J.I. Warrick will be required to determine the precise mechanism underly- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, ing the contribution of the DUOX2Y1203H missense variant to computational analysis): D.V. Bann, Q. Jin, J.R. Broach, G.S. Gerhard increased susceptibility to nonmedullary thyroid cancer. Writing, review, and/or revision of the manuscript: D.V. Bann, K.R. Houser, Although it is possible that the Y1203H mutation alters J.I. Warrick, M.J. Baker, J.R. Broach, G.S. Gerhard, D. Goldenberg the interaction between DUOX2 and DUOXA2, several obser- Administrative, technical, or material support (i.e., reporting or organizing vations argue against this hypothesis. First, the interaction data, constructing databases): D.V. Bann, K.R. Houser, D. Goldenberg Study supervision: J.R. Broach, D. Goldenberg between DUOX2 and DUOXA2 is mediated by the N- Other (provided cancer genetic counseling to family members regarding terminal extracellular A-loop of DUOX2, which is remote from risks, benefits, and limitations of genetic testing; developed the family Y1203 (37). In addition, the location of Y1203 within a pedigree): M.J. Baker transmembrane domain (37) suggests that Y1203H is not likely to participate in protein–protein interactions. Finally, protein– Acknowledgments protein interactions between DUOX2 and DUOXA2 are This project is funded, in part, under a Pennsylvania Department of Health required for the maturation and function of DUOX2 (37). Our Tobacco CURE grant (#4100072562 to D. Goldenberg). The Department fi data indicate that DUOX2Y1203H is enzymatically active, there- speci cally disclaims responsibility for any analysis, interpretation, or by providing indirect evidence for an intact interaction between conclusions. DUOX2Y1203H and DUOXA2. The identification of DUOX2 as a potential causes of FNMTC The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked led us to investigate whether DUOX2 dysregulation may also play advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate a role in sporadic PTC. The rs965513 variant is a common this fact. polymorphism associated with 3.1-fold increased risk of devel- oping thyroid cancer in homozygous individuals (6). We found Received March 6, 2019; revised May 3, 2019; accepted September 3, 2019; that 23% of the patients with sporadic PTC analyzed in this study published first September 9, 2019.

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Genetic Variants Implicate Dual Oxidase-2 in Familial and Sporadic Nonmedullary Thyroid Cancer

Darrin V. Bann, Qunyan Jin, Kathryn E. Sheldon, et al.

Cancer Res 2019;79:5490-5499. Published OnlineFirst September 9, 2019.

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