DUOX2 Variant Causes Hereditary Thyroid Cancer

Author Manuscript Published OnlineFirst on September 9, 2019; DOI: 10.1158/0008-5472.CAN-19-0721 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Running Title: DUOX2 Variant Causes Hereditary Thyroid Cancer

Genetic Variants Implicate Dual Oxidase-2 (DUOX2) in Familial and Sporadic Non- Medullary Thyroid Cancer

Darrin V. Banna,b, Qunyan Jinc, Kathryn E. Sheldonb, Kenneth R. Houserd, Lan Nguyenb, Joshua I. Warricke, Maria J. Bakerf, James Broachb,d, Glenn S. Gerhardc, David Goldenberga

aDepartment of Otolaryngology – Head & Neck Surgery, The Pennsylvania State University, College of Medicine, Hershey, PA 17033 USA bInstitute for Personalized Medicine, The Pennsylvania State University, College of Medicine, Hershey, PA 17033 USA cLewis Katz School of Medicine at Temple University, Philadelphia, PA 19140 USA dDepartment of Biochemistry and Molecular Biology, The Pennsylvania State University, College of Medicine, Hershey, PA 17033 USA eDepartment of Pathology, The Pennsylvania State University, College of Medicine, Hershey, PA 17033 USA fDepartment of Hematology/Oncology, The Pennsylvania State University, College of Medicine, Hershey, PA 17033 USA

Keywords: Familial non-medullary thyroid cancer, hereditary thyroid cancer, papillary thyroid cancer, dual oxidase-2, reactive oxygen species

Financial Support: This work was supported, in part, by a grant with the Pennsylvania Department of Health using Tobacco CURE funds to DG.

Conflict of Interest: The authors declare no competing conflict of interest.

Word Count: 4,693 Figures: 3 Tables: 1, plus 2 supplemental

Corresponding Author and Request for Reprints: David Goldenberg MD, FACS The Pennsylvania State University College of Medicine Department of Surgery 500 University Drive, H091 Hershey, PA 17033-0850 Phone: (717) 531–8946 Fax: (717) 531–6160 E-mail: [email protected]

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 9, 2019; DOI: 10.1158/0008-5472.CAN-19-0721 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Highly penetrant hereditary thyroid cancer manifests as familial nonmedullary thyroid cancer

(FNMTC), while low-penetrance hereditary thyroid cancer manifests as sporadic disease and is associated with common polymorphisms, including rs965513. Whole-exome sequencing of an

FNMTC kindred identified a novel Y1203H germline Dual Oxidase-2 (DUOX2) mutation.

DUOX2Y1203H is enzymatically active and increased production of reactive oxygen species.

Furthermore, sporadic thyroid cancer patients homozygous for rs965513 demonstrated higher

DUOX2 expression than heterozygous or homozygous negative patients. These data suggest that dysregulated hydrogen peroxide metabolism is a common mechanism by which high- and low- penetrance genetic factors increase thyroid cancer risk.

Significance

This study provides novel insights into the genetic and molecular mechanisms underlying familial and sporadic thyroid cancers.

Introduction

Thyroid cancer is the most common endocrine malignancy in the United States, resulting

in 62,450 new diagnoses and 1,950 deaths in 2015 (1). Compared to other common cancers,

thyroid cancer displays a high degree of heritability with first-degree relatives having a 5.5-fold

increase in relative risk of disease (2). This increased thyroid cancer risk results from both high

penetrance rare variants and low penetrance common variants (3-8). Hereditary cancer

syndromes associated with medullary thyroid cancer have been well described, however the

genetic lesions associated with nonmedullary thyroid cancer heritability are less well defined.

Familial nonmedullary thyroid cancer (FNMTC) is defined as thyroid cancer of follicular cell

origin in two or more first-degree relatives in the absence of other cancer predisposition

syndromes (9) and is thought to account for 3 – 10% of all thyroid cancers (10). FNMTC

families display a mix of benign and malignant thyroid tumors with an earlier age of onset and a

higher incidence of multifocal disease compared to patients with sporadic thyroid cancer (10,11).

Germline mutations in HABP2, SRRM2, and FOXE1 have been reported in FNMTC kindreds

(3-5), however whether all of these mutations truly cause FNMTC remains controversial (12-14).

In contrast to FNMTC, low-penetrance hereditary thyroid cancer has been associated

with multiple common single nucleotide polymorphisms (SNPs) (6-8). The rs965513[A] allele,

which confers the greatest relative risk for the development of thyroid cancer (6,7), resides

within an enhancer element controlling expression of FOXE1 (15,16), a thyroid-specific

transcription factor that regulates several genes including thyroid peroxidase (TPO), thyroglobulin (TG), the sodium-iodide symporter (SLC5A5), and dual oxidase-2 (DUOX2) (17).

The risk allele is associated with decreased expression of FOXE1 and siRNA knockdown of

FOXE1 causes altered expression of DUOX2, TPO, TG, and SLC5A5 in vitro (17).

Using whole-exome sequencing, we identified a novel Y1203H germline DUOX2 mutation in an FNMTC kindred. DUOX2 generates hydrogen peroxide (H2O2), which is used by

TPO to iodinate tyrosyl residues on TG for thyroxine (T4) and 3,3’,5-triiodothyronine (T3) synthesis, and loss-of-function mutations in DUOX2 cause congenital hypothyroidism (18). We

report that DUOX2Y1203H is functionally active and is highly expressed in normal thyroid tissue, potentially contributing to increased tumorigenesis. Strikingly, DUOX2 expression is increased in patients with rs965513[A] homozygosity, consistent with a model in which DUOX2-mediated dysregulation of H2O2 metabolism underlies both high- and low-penetrance hereditary thyroid

cancers.

Materials & Methods

Subjects were recruited at the Penn State College of Medicine. The study was approved

by the Penn State College of Medicine Institutional Review Board. Written informed consent

was obtained from all participants whose DNA and tumor tissues were analyzed.

Nucleic acid extraction, whole exome capture, and next-generation sequencing

Germline DNA was extracted from whole blood or saliva using a Qiagen QIAsymphony

nucleic acid isolation robot. Qiagen AllPrep DNA/RNA Mini Kits (Qiagen, CA) were used to

extract nucleic acids from tumor and normal tissue stored in RNAlater at -80˚C (ThermoFisher,

MA) or liquid nitrogen. Additional details are provided in Supplemental Methods.

Genomic DNA was sheared into ~200 bp fragments and processed into Illumina

compatible sequencing libraries with a PrepX ILM DNA Library Kit (Wafergen, Fremont, CA)

on an Apollo 324 instrument (Wafergen, Fremont, CA). Sheared DNA (500 to 1,000 ng) was

5 processed into Illumina compatible sequencing libraries with a PrepX ILM DNA Library Kit

(Wafergen, Fremont, CA) on an Apollo 324 instrument (Wafergen, Fremont, CA). Libraries were uniquely barcoded, amplified, and subjected to multiplex SeqCap EZ Exome V3.0 (Roche,

Basel, Switzerland) hybridization and capture. Libraries were sequenced on an Illumina 2500

(Illumina Inc., CA) using 2x100 bp reads in high-output mode. Sequence data have been deposited at the European Genome-phenome Archive (EGA), which is hosted by the EBI and the

CRG, under accession number EGAS00001002258. Further information about EGA can be

found on https://ega-archive.org "The European Genome-phenome Archive of human data

consented for biomedical research"

(http://www.nature.com/ng/journal/v47/n7/full/ng.3312.html). Additional details regarding

library preparation and sequencing are provided in Supplemental Methods.

Sequencing data pipeline and variant filtering

Next-generation sequencing data were processed according to Genome Analysis Tool Kit

(GATK) best practices recommendations using GATK v. 2.7.4 (19-21). Variants within the

exome capture region +/- 100 bp were identified using the GATK Haplotype Caller module v3.3.0 (20,21). Additional details are provided in Supplemental Methods. Germline variants were

filtered using Golden Helix SNP and Variation Suite v8.1.5 (Golden Helix, Inc., MT) according

to the following criteria: 1) segregation with an autosomal dominant mechanism of inheritance;

2) SNPs produced a non-synonymous variation in a defined exon; 3) SNPs were present in

<0.5% of the population encompassed by the 1,000 Genomes Project Phase 1, the dbSNP build

137 common variants database (http://www.ncbi.nlm.nih.gov/SNP) (22), the NHLBI GO Exome

Sequencing Project build 138 database (23), and the Exome Aggregation Consortium (ExAC) database version 0.3.1 (24); and 4) SNPs had a variant quality score >20 and read depth >10.

We next filtered for variants in genes that had a previously known function reported in

PubMed and for those highly expressed in thyroid tissue. Tissue-specific gene expression data

was derived from Illumina Body Map 2.0, GTEx, and Uhlen’s lab experiments in the EMBL-

EBI Expression Atlas Database (25-27). Variants were retained if they occurred within a gene

differentially expressed in thyroid tissue across all three data sets. Differential expression was

defined by 퐸푖 > 1.5(푄3 − 푄1) + 푄3 where 퐸푖 represents expression of gene i in thyroid tissue,

푄3 represents the third quartile expression value for gene i across all tissues, and 푄1 represents

the first quartile gene expression value for gene i across all tissues. Variants occurring in genes

where the raw expression value was less than the mean 푄1 value for all genes in thyroid tissue

across all three databases were also excluded. PolyPhen-2 (28) scores were used to identify

variants with potential functional impact, and variants with a score <0.3 were excluded. For the

remaining variants, literature searches were conducted to identify any potential function for

impacted genes in the thyroid gland; only genes directly implicated in thyroid gland physiology

were included. Further testing required that candidate SNPs be absent in an unaffected and

unrelated spouse and present in an additional affected family member.

Somatic mutation detection

Somatic mutations in tumor exomes were detected using MuTect v1.1.7 (29). To reduce

the incidence of false-positive mutation calls due to sequencing artifacts, alignment errors, and

rare germline mutations the -panelofnormals flag was implemented as described (30) using white

blood cell whole-exome sequence data from 50 patients with no known hematologic malignancy.

Details regarding the “panel of normals” can be found in supplemental methods. Filtered VCFs

were annotated using TabAnno v2.13 (31) and somatic mutations were confirmed by visual

inspection in Integrative Genomics Viewer (32).

Protein modeling and alignment

Modeling of the DUOX2 peroxidase domain (NCBI accession NP_054799.4) was

performed using SWISS-MODEL (33). Amino acid sequences of DUOX2 or the orthologous

protein were obtained for the following species: H. sapiens (NCBI accession NP_054799.4), P. troglodytes (NCBI accession XP_009427327.1), M. mulatta (NCBI accession XP_014997623.1),

C. lupus familiaris (NCBI accession XP_013964824.1), B. taurus (NCBI accession

XP_010807708.1), M. musculus (NCBI accession NP_808278.2), R. norvegicus (NCBI

accession NP_077055.1), D. melanogaster (NCBI accession NP_001259968.1), and C. elegans

(NCBI accession NP_490684.1). Protein alignment was performed using Clustal Omega (34).

PCR, Sanger sequencing, and targeted deep sequencing

Sanger sequencing was used to verify variants identified by next generation exome

sequencing. PCR amplification of regions spanning DUOX2Y1203, rs965513, BRAFV600,

KRASQ61, HRASQ61, and NRASQ61 was conducted using primers shown in Supplemental Table

1. Details regarding PCR conditions are described in Supplemental Methods. Sanger sequencing

was conducted by GENEWIZ LLC. (NJ). Mutation status was determined by visual inspection of

fluorescence trace files of capillary electrophoresis. Targeted deep sequencing libraries of gel-

purified BRAFV600 and KRASQ61 PCR products were constructed using the KAPA hyper prep kit

(KAPA Biosystems, MA). Libraries were sequenced on an Illumina MiSeq instrument

8 generating 2x250 bp reads. Adapter sequences were trimmed and reads were aligned to the hg19 reference genome using the FASTQ Toolkit and BWA Aligner apps on the Illumina BaseSpace website (http://basespace.illumina.com). Minor allele frequencies were determined by manual inspection of BAM files in Integrative Genomics Viewer (32).

Quantitative reverse transcription PCR

Extracted RNA was DNAse treated using an Ambion TURBO DNA-free kit

(ThermoFisher, MA) and reverse transcribed using an Applied Biosystems High-Capacity cDNA

Reverse Transcription Kit (ThermoFisher, MA) according to the manufacturer’s instructions.

Quantitative PCR was performed using Applied Biosystems (ThermoFisher, MA) TaqMan Gene

Expression Master Mix and TaqMan Probes targeting FOXE1 (Cat. No.Hs00916085-S1),

DUOX2 (Cat. No.Hs00204187-m1), TPO (Cat. No.Hs0892518-m1), TG (Cat. No.Hs00794359-

m1), and GAPDH (Cat. No. Hs02758991-G1). Reactions were analyzed on an Applied

Biosystems QuantStudio 12K Flex Instrument (ThermoFisher, MA). Expression of each target

was determined as fold expression relative to GAPDH averaged over three independent

replicates. Differences in gene expression relative to rs965513 genotype were computed using

the Kruskal-Wallis Rank Sum Test as implemented by the “stats” package in R v3.2.2 (35).

Plasmids and mutagenesis

The vectors EX-NEG-M02, human DUOX2 (EX-E1601-M02), and human DUOXA2

(EX-H0633-M02) were purchased from Genecopoeia (MD). The DUOX2Y1203H expression

vector was prepared from the wild-type DUOX2 cDNA using the QuickChange lightning site-

directed mutagenesis kit (Agilent, CA) with forward primer 5'-

9 gggaggcgaagacatgcatgatggccaggac-3' and reverse primer 5'-gtcctggccatcatgcatgtcttcgcctccc-3' according to the manufacturer’s protocols. Following mutagenesis the construct was confirmed by bi-directional Sanger sequencing.

Cell culture and stable transfections

Cos7 (CRL1651, ATCC, VA) cells were cultured in Dulbecco's modified Eagle's medium

0 (ATCC, VA) with 10% fetal bovine serum (ATCC, VA) under 5% CO2/95% air at 37 C. To produce stable cell lines Cos7 cells were transfected with wild-type human DUOXA2 in a CMV driven expression vector (pReceiver-M02) or empty vector using Lipofectamine 3000 (Thermo

Fisher, MA) transfection reagent according to the manufacturer's protocol. Forty-eight hours after transfection cells were re-plated in medium with Geneticin (G418) (Thermo Fisher, MA)

(500 μg/ml) for selection. After 2 weeks of selection, cells were pooled, expanded, and maintained with Geneticin (G418) (250 μg/ml).

Reactive oxygen species measurements

Multiplex ROS-Glo H2O2 Assay (Promega, WI) and Amplex Red® Hydrogen

Peroxide/Peroxidase Assay (ThermoFisher Scientific, MA), were used to measure extracellular

H2O2,, while intracellular ROS were measured using a DCFDA ROS Detection Assay kit

(AbCam, UK). For all assays, Cos7 cells stably expressing DuoxA2 were plated in 96-well plates and grown to 70%-80% confluence according to the manufacturer’s protocol. Cells were then transiently transfected with the EX-NEG-M02 empty vector, DUOX2 vector, or the

DUOX2Y1203H vector using Lipofectamine 3000 (ThermoFisher Scientific, MA) according to the manufacture’s protocol. Twenty-four hours after transfection the ROS and cell viability assays

10 were performed according to the manufacturer’s instructions. Relative luminescence was determined using a SpectraMax I3 Multiplate reader (Molecular Devices, CA). All experiments were performed in triplicate. ROS production for each transfection condition was standardized to the empty vector transfection negative control. Statistical differences in ROS production were calculated using the Students T-test as implemented in Microsoft Excel (Microsoft Corporation,

WA).

Similar protein expression across experiments was confirmed using semi-quantitative

Western blotting of cells transfected in parallel. Briefly, cell extracts were prepared in RIPA buffer (Sigma, MO) supplemented with protease inhibitor cocktail (Sigma, MO) and protein concentrations were determined by the BCA method (Pierce, IL). Equal quantities of protein input (30 μg) were separated by 4-12% Nupage Tris-Bis gel electrophoresis with SeeBlue Plus 2

Protein Standard (Invitrogen, CA) under reducing conditions and transferred to an Immobilon-P transfer membrane (EMD Millipore, MA). Immunoblots were incubated DUOX2 S-12

(MABN787; EMD Millipore, MA) diluted 1:1000, GAPDH diluted 1:1000 (sc-98501; Santa

Cruz Biotechnology, CA) and DUOXA2 diluted 1:100 (sc-98501; Santa Cruz Biotechnology,

CA). Blots were processed using a ScanLater kit with appropriate secondary antibodies according to the manufacturer’s instructions (Molecular Devices, LLC) and quantified using

ImageJ (36).

Immunohistochemistry

Slides were prepared from 5 m sections of formalin-fixed paraffin-embedded tissue.

Representative sections were stained with hematoxylin and eosin and fitted with glass coverslips for imaging. Remaining samples were deparaffinized using standard procedures and antigen

retrieval was performed by incubation in 10 mM sodium citrate, pH 6.0 with 1 mM EDTA at

95˚C for 20 minutes. Samples were incubated with mouse anti-DUOX2 S-12 monoclonal

antibody (0.5 mg/mL, diluted 1:1,000) (MABN787; EMD Millipore, MA) at 4˚C overnight.

Slides were then incubated with undiluted ImmPRESS goat anti-mouse-HRP secondary antibody

(Vector Laboratories, CA) for 30 minutes at room temperature, washed, and developed with

diaminobenzidine (DAB) (Vector Laboratories, CA). Slides were counterstained with

Hematoxylin prior to being fitted with glass coverslips for imaging. Slides were reviewed by a

board-certified Head and Neck Pathologist (JIW).

Results

Identification of a novel missense mutation associated with FNMTC

The proband (Fig. 1A, patient III.2) presented with multifocal micropapillary thyroid

carcinoma at 46 years of age. She had an extensive family history of thyroid cancer. The

proband’s maternal grandmother (Fig. 1A, patient I.2), who ultimately succumbed to the disease,

had three children (II.2, II.3, and II.4) all of whom were diagnosed with thyroid cancer.

Individual II.2 has been treated for highly aggressive, recurrent PTC at our institution (Fig. 1A).

Individuals II.1 and II.2 had four children, all of whom were affected: two with papillary thyroid

cancer (proband and III.4), and two with nodular thyroid disease (III.3 and III.5) (Fig. 1A). The

disease status of individuals III.6, III.7, and III.8 is not known. The proband (III.2) has two

children (IV.1 and IV.2), one of whom (IV.1) was diagnosed with a benign thyroid nodule by

ultrasound examination at age 19 (Fig. 1A). Importantly, no patient in this pedigree exhibited

clinical symptoms of hypothyroidism. The pre-thyroidectomy thyroid stimulating hormone

(TSH) level for the proband patient was 4.4 IU/mL (reference range 0.35 – 5.5 IU/mL),

12 although preoperative TSH levels were not available for the other individuals in the study. This

pedigree is consistent with autosomal dominant hereditary papillary thyroid cancer (Fig. 1A).

Germline DNA from five affected individuals (Fig. 1A, dashed lines) was subjected to

whole-exome sequencing. After quality, frequency, segregation, and functional filtering,

missense variants were identified in three genes: DUOX2, IFT140, and WDR72 (Table 1).

Because of its central role in thyroid physiology we focused our analysis on DUOX2. A single,

heterozygous A to G variant at chr15:45,389,898, predicted to cause a Y1203H substitution in

DUOX2 (Table 1), was identified in all affected individuals (Fig. 1A). Sanger sequencing

confirmed the presence of the variant in all affected individuals but not in an unaffected family

member (Fig. 1A, patient III.1). The probability of this mutation occurring in all affected family

members is approximately 1.6%. Analysis of the dbSNP database (22) revealed that the

prevalence of this mutation in the general population is approximately 1/138,000 individuals,

indicating that DUOX2Y1203H is an extremely rare mutation. No hereditary cancer predisposition

syndromes known to cause thyroid cancers were detected in affected individuals (Supplemental

Table 2). Furthermore, all sequenced individuals were negative for the HABP2G534E,

FOXE1A248G, and SRRM2S346E polymorphisms previously implicated in FNMTC (Supplemental

Table 2) (3,4).

The DUOX2 protein consists of an extracellular N-terminal peroxidase domain; seven

transmembrane domains containing four invariant histidines, which may be coordination sites for

two nonidentical prosthetic heme moieties (37); two EF-Hand calcium binding domains; and

intracellular C-terminal FAD- and NADPH-binding domains (Fig. 1B). Expression, proper localization and function of DUOX2 is dependent on its cognate maturation factor, DUOXA2

(Fig. 1B), which interacts with the extracellular ‘A’ loop of DUOX2 (37). The Y1203H mutation

13 is located within the fifth transmembrane domain, which is distant from the enzymatic active site and the DUOX2A interaction domain (Fig. 1B), but is highly conserved across mammalian species (Fig. 1C).

Functional analysis of DUOX2Y1203H

Previous reports have described DUOX2 mutations, predominantly in the extracellular peroxidase domain or the intracellular calcium-binding domain, resulting in loss of function and congenital hypothyroidism (37). We therefore tested whether the DUOX2Y1203H variant disrupts

the H2O2 and reactive oxygen species (ROS) generating capacity of the enzyme. Because

DUOX2 expression and H2O2 requires co-expression of DUOXA2 (37), we generated a Cos7

cell line that stably expressed human DUOXA2 (Cos7-DUOXA2). After transfection of Cos7-

DUOXA2 cells with empty vector, wild type DUOX2, or DUOX2Y1203H, extracellular H2O2

production was measured using independent RosGlo and Amplex Red® assays, while

intracellular global ROS levels were measured using the DCFDA ROS detection assay (Fig. 1D).

Wild type DUOX2 and DUOX2Y1203H significantly increased extracellular and intracellular H2O2 and ROS levels relative to empty vector controls. The Amplex Red® assay demonstrated increased extracellular H2O2 production by DUOX2Y1203H compared to wild-type DUOX2.

Western blotting confirmed similar expression of wild-type and mutant DUOX2 in each

experiment (Fig. 1E). Moreover, wild type and mutant DUOX2 were expressed at low levels in

Cos7 cells stably transfected with empty vector (Fig. 1E, Cos7-EV), confirming that

DUOX2Y1203H expression is dependent upon DUOXA2 as expected (37). These data demonstrate

that DUOX2Y1203H contains a functional peroxidase domain capable of producing H2O2 and

suggest that DUOX2Y1203H increases extracellular reactive oxygen species compared to wild-type

DUOX2.

DUOX2 expression in normal thyroid and thyroid tumor tissue

We next sought to determine the effect of DUOX2Y1203H on normal thyroid and thyroid

tumor histology by comparing tissue samples from the proband patient (Fig. 1A, patient III.2)

and a patient with sporadic thyroid cancer. Upon H&E staining, non-tumor tissue from the

proband patient was normal in appearance (Fig. 2A) and was similar to non-tumor thyroid tissue

from the sporadic thyroid cancer patient (Fig. 2B). Histological examination of the proband’s

thyroid gland revealed two distinct tumors (Fig. 2A): a 7mm tumor with follicular variant

papillary thyroid cancer (FV-PTC) architecture (Fig. 2A, Micro-PTC 1), and a 4mm tumor with

classical papillary architecture (PTC) (Fig. 2A, Micro-PTC 2). A single tumor with classical

papillary architecture was identified in the thyroid gland extirpated from the sporadic thyroid

cancer patient (Fig. 2B).

DUOX2 is highly expressed in thyroid follicular cells (25,38). To determine the effect of

the Y1203H substitution on DUOX2 expression we performed immunohistochemistry to

compare DUOX2 expression between the proband and the sporadic thyroid cancer patient.

Antibody specificity was confirmed by Western blotting (Fig. 1E). Thyroid follicular cells

stained strongly for DUOX2 in normal tissue from the proband (Fig. 2A, -DUOX2) and an

unrelated sporadic thyroid cancer patient with wild-type DUOX2 (Fig. 2B, -DUOX2). The

proband’s MicroPTC-1 lesion exhibited strong DUOX2 staining, although expression was

grossly reduced in MicroPTC-2 (Fig. 2A), suggestive of clonally distinct tumors arising within a

single individual. The tumor isolated from the sporadic PTC patient exhibited DUOX2

15 expression similar to adjacent normal thyroid tissue (Fig. 2B). Together, these data indicate that

DUOX2Y1203H heterozygosity does not dramatically reduce DUOX2 expression, although

expression of DUOX2 may vary depending upon the differentiation pattern of thyroid tumors.

To determine whether DUOX2Y1203H was associated with DNA mutagenesis in the

thyroid gland we profiled the mutational spectrum of tumor tissue and adjacent normal tissue

from the proband and the sporadic thyroid cancer patient using whole exome sequencing. The

MicroPTC-1 tumor from the proband was positive for the KRASQ61R oncogenic driver mutation

while the MicroPTC-2 tumor was positive for the BRAFV600E driver mutation (Fig. 2A). The

mutual exclusivity of oncogenic driver mutations in the two tumors was confirmed by Sanger

sequencing and targeted next-generation sequencing of the mutated regions to a depth >15,000X

(Fig. 2A). Sequence analysis of the sporadic tumor revealed a single BRAFV600E mutation (Fig.

2B). The exclusivity BRAFV600E oncogenic driver mutation was confirmed by targeted next-

generation sequencing (Fig. 2B).

The rs965513 thyroid cancer risk polymorphism is associated with increased DUOX2

expression

The mechanism through which the rs965513 polymorphism influences risk of non-

medullary thyroid cancer is not known. We hypothesized that altered expression of FOXE1, and

consequently DUOX2, in patients with the rs965513 polymorphism may underlie sporadic

thyroid cancer susceptibility. To test this hypothesis we genotyped 64 thyroid cancer patients for

the rs965513 allele using Sanger sequencing and quantified expression of FOXE1, DUOX2, and

other thyroid-specific genes in normal thyroid tissue by quantitative PCR. The A/G genotype

was the most common (n = 32, 50%) followed by the G/G genotype (n = 17, 27%) and the A/A

16 genotype (n = 15, 23%). Expression of FOXE1 was lower in the high-risk A/A group compared to the G/G and A/G genotypes, although this difference did not reach statistical significance (Fig.

3A). Higher expression of DUOX2 (P = 0.033), TPO (P = 0.031), TG (P = 0.029), and SLC5A5

(P = 0.014) were found in the A/A group (Fig. 3B-E), consistent with decreased FOXE1 promoter activity (8).

Discussion

Few genes have been identified as bona fide causes of FNMTC, partially due to the low

prevalence of familial disease. The estimated prevalence of thyroid cancer in the United States is

0.14% (1) with FNMTC accounting for 3-10% of cases (10). Thus only 0.0042 – 0.014% of

individuals in the general population are likely to be affected by FNMTC in their lifetime. Our

analysis of a single highly penetrant pedigree using exome sequencing identified a novel

germline Y1203H missense variant in DUOX2 that segregated as an autosomal dominant thyroid

cancer phenotype. The DUOX2Y1203H variant appears to be a very rare mutation that has been

identified in only 1/138,000 individuals in the dbSNP database (22), consistent with its role as a

causative mutation in a rare condition such as familial non-medullary thyroid cancer.

Other germline variants, including HABP2G534E, SRRM2S346F, and FOXE1A248G (3-5) have been reported to cause FNMTC. The prevalence of HABP2G534E SNP at 1-5% (13,14,22,39) and

FOXE1A248G at 5.3/1,000 (22) are not consistent with the predicted prevalence of FNMTC at

1/~10,000 individuals in the general population (10). The SRRM2S346F polymorphism is more

rare with a predicted prevalence of 6/10,000 (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 data sets, consistent

with the apparent rarity of DUOX2Y1203H (22). Furthermore, 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 carcinogenesis 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 compared with sporadic thyroid cancer patients (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 towards 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 extracelluar ROS (ROSGlo and Amplex Red) indicate that DUOX2Y1203H increases

extracellular ROS levels compared to wild-type DUOX2, while measurement of intracellular

ROS using DCFDA demonstrated similar ROS production between wild-type and mutant

DUOX2. These data suggest that the DUOX2Y1203H mutation may result in dysregulated ROS production at the plasma membrane, however further studies are required to evaluate this possibility. Although the differences in ROS production that we observed were small and measured over a short time period (24 hours), changes in ROS metabolism that persist over many

years may have cumulative biological effects that contribute to thyroid tumorigenesis, which

occurs over the course of decades.

Homozygous or compound heterozygous mutations that compromise DUOX2 enzymatic

activity and result in congenital hypothyroidism are found primarily in the N-terminal

extracellular peroxidase-like domain and in the EF hand domains (18,38,44). By contrast, the

tyrosine at position 1203 resides in the fifth transmembrane domain. To date, only three DUOX2

transmembrane mutations have been identified in association with congenital hypothyroidism, all

in the context of compound heterozygosity for other damaging mutations (45-47). There was no

evidence of clinical hypothyroidism in the kindred we analyzed.

The transmembrane location of the Y1203H mutation raises the possibility that

DUOX2Y1203H is mislocalized, resulting in dysregulated H2O2 metabolism. Indeed, other mutations in DUOX2 have been described that result in mislocalization of an enzymatically active protein to an intracellular compartment and presumed enhanced degradation (44,48).

2+ However, it is also possible that because H2O2 generation is Ca - dependent (49), enzyme

function may be enhanced by increased calcium binding or availability in different subcellular

locations. Increased oxidative damage from H2O2 produced by DUOX2Y1203H in vivo may also

result from disruption of the physiologic colocalization of DUOX2 and TPO at the apical plasma

membrane, although free H2O2 can lead to DUOX2 inactivation (50). It is notable that the

Y1203H mutation is near two transmembrane heme moieties that coordinate via histidine

19 residues (37). The Y1203H substitution may therefore impact heme binding, although the effect of altered heme binding on protein function is not known. Future studies will be required to determine the precise mechanism underlying the contribution of the DUOX2Y1203H missense

variant to increased susceptibility to non-medullary thyroid cancer.

While it is possible that the Y1203H mutation alters the interaction between DUOX2 and

DUOXA2, several observations argue against this hypothesis. First, the interaction between

DUOX2 and DUOXA2 is mediated by the N-terminal extracellular A-loop of DUOX2, which is

remote from Y1203 (37). In addition, the location of Y1203 within a transmembrane domain

(37) suggests that Y1203H not likely to participate in protein-protein interactions. Finally,

protein-protein interactions between DUOX2 and DUOXA2 are required for the maturation and

function of DUOX2 (37). Our data indicate that DUOX2Y1203H is enzymatically active, thereby

providing indirect evidence for an intact interaction between DUOX2Y1203H and DUOXA2.

The identification of DUOX2 as a candidate FNMTC gene led us to investigate whether

DUOX2 dysregulation may also play a role in sporadic PTC. The rs965513 variant is a common

polymorphism associated with 3.1-fold increased risk of developing thyroid cancer in

homozygous individuals (6). We found that 23% of the sporadic PTC patients analyzed in this

study were homozygous for rs965513, more than double the expected 11 - 12% prevalence of

rs965513 homozygosity in the general population (6,22). We found that individuals homozygous

for rs965513 expressed higher levels of DUOX2, TPO, TG, and SLC5A5 (Fig. 3) compared to

heterozygotes or homozygous-reference individuals. These data are consistent with a potential

mechanism for the rs965513 allele to increase thyroid cancer susceptibility via enhanced

DUOX2 expression and H2O2 production. Similar mechanisms may therefore underlie highly

20 penetrant FNMTC and some cases of PTC caused by low-penetrance risk factors including rs965513.

We note the significant intra-group variability in gene expression. Given that each data point represents a mean of three independent experiments, it is likely that there is true biological variability in gene expression among the population, although the underlying reasons not known.

Possibilities for variability in thyroid-specific gene expression may include undiagnosed thyroid pathology, such as hypothyroidism or autoimmune thyroid disease, and environmental effects.

In summary, we used an unbiased approach to identify a novel germline mutation in

DUOX2 associated with highly penetrant FNMTC. Combined with evidence that the FOXE1 rs965513 polymorphism also increases DUOX2 expression, our data suggest that dysregulation of proteins involved in H2O2 metabolism may be a common mechanism underlying common high- and rare low-penetrance genetic factors that increase thyroid cancer susceptibility. This

observation has translational implications for screening, chemoprevention, and development of

therapeutics.

Acknowledgments

This project is funded, in part, under a Pennsylvania Department of Health Tobacco CURE grant to DG (#4100072562). The Department specifically disclaims responsibility for any analysis, interpretation, or conclusions.

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Tables

Table 1. Whole exome sequencing variant filtering protocol Filtering step Mean coverage depth Number of variants 1) Variants Identified  Patient II.2 107.3X 644,267  Patient III.2 68.1X 541,180  Patient III.3 90.4X 608,064  Patient III.4 112.7X 1,728,519  Patient III.5 94.4X 1,826,547 2) Heterozygous Variants Present in All Affected Family Members 33,234 3) Non-synonymous variants in protein coding exons 1,560 4) Variants in <0.5% of population represented by Exome Variant 838 Server, dbSNP, and 1,000 Genomes Databases 5) Genotype quality >20 and read depth >20 823 6) Variant in a highly-expressed gene that is differentially expressed in 6 thyroid tissue 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; S953Vfs. No evidence for specific function in thyroid, may be involved in parathyroid function

Figure Legends

Figure 1. Familial non-medullary 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 acids

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 standard error of

the mean. Data were normalized to the baseline established by EV transfection, which was set to

a value of 1. (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.

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 immunohistochemistry with -DUOX2 antibodies (α-

DUOX2). Targeted next-generation sequencing to a depth >15,000X 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 H&E or were subjected to immunohistochemistry with α-DUOX2 antibodies.

The exclusivity of this mutation within tumor tissue was confirmed by targeted next-generation

sequencing to a depth of >600X. 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.

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 (A) FOXE1, (B) DUOX2, (C) TPO, (D)

TG, and (E) SLC5A5 and are expressed as log fold expression relative to GAPDH controls. Each

31 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 1.5x the interquartile range. *P < 0.05.

Genetic Variants Implicate Dual Oxidase-2 (DUOX2) in Familial and Sporadic Non-Medullary Thyroid Cancer

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

Cancer Res Published OnlineFirst September 9, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-0721

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