Functional Zebrafish Studies Based on Human Genotyping Point to Netrin-1 as a Link Between Aberrant...

Functional Zebrafish Studies Based on HumanGenotyping Point to Netrin-1 as a Link BetweenAberrant Cardiovascular Development and ThyroidDysgenesis

Robert Opitz,* Marc-Philip Hitz,* Isabelle Vandernoot, Achim Trubiroha,Rasha Abu-Khudir, Mark Samuels, Valérie Désilets, Sabine Costagliola,*Gregor Andelfinger,* and Johnny Deladoëy*

Institute of Interdisciplinary Research in Molecular Human Biology (R.O., I.V., A.T., S.C.), Université Librede Bruxelles, 1070 Brussels, Belgium; Research Center of Centre Hospitalier Universitaire Sainte-Justine(M.-P.H., I.V., R.A.-K., M.S., G.A., J.D.), Department of Pediatrics, Université de Montréal, Montréal,Québec, Canada H3T 1C5; and Department of Medical Genetics (V.D.), Centre Hospitalier Universitairede Sherbrooke, Sherbrooke, Canada J1H 1P8

Congenital hypothyroidism caused by thyroid dysgenesis (CHTD) is a common congenital disorderwith a birth prevalence of 1 case in 4000 live births, and up to 8% of individuals with CHTD haveco-occurring congenital heart disease. Initially we found nine patients with cardiac and thyroidcongenital disorders in our cohort of 158 CHTD patients. To enrich for a rare phenotype likely tobe genetically simpler, we selected three patients with a ventricular septal defect for molecularstudies. Then, to assess whether rare de novo copy number variants and coding mutations incandidate genes are a source of genetic susceptibility, we used a genome-wide single-nucleotidepolymorphism array and Sanger sequencing to analyze blood DNA samples from selected patientswith co-occurring CHTD a congenital heart disease. We found rare variants in all three patients, andwe selected Netrin-1 as the biologically most plausible contributory factor for functional studies.In zebrafish, ntn1a and ntn1b were not expressed in thyroid tissue, but ntn1a was expressed inpharyngeal arch mesenchyme, and ntn1a-deficient embryos displayed defective aortic arch arteryformation and abnormal thyroid morphogenesis. The functional activity of the thyroid in ntn1a-deficient larvae was, however, preserved. Phenotypic analysis of affected zebrafish indicates thatabnormal thyroid morphogenesis resulted from a lack of proper guidance exerted by the dysplasticvasculature of ntn1a-deficient embryos. Hence, careful phenotyping of patients combined withmolecular and functional studies in zebrafish identify Netrin-1 as a potential shared genetic factorfor cardiac and thyroid congenital defects.

Congenital hypothyroidism caused by thyroid dysgen-esis (CHTD) is a common congenital disorder with a

birth prevalence of 1 case in 4000 live births (1), and up to8% of individuals with CHTD have co-occurring congen-

ital heart disease (CHD) (2). Incomplete migration of thethyroid resulting in ectopic tissue (sublingual thyroid) isthe most common thyroid developmental defect (up to80%), with athyreosis and orthotopic thyroid hypoplasia

* R.O., M.-P.H., S.C., G.A., and J.D. contributed equally to this work.

Abbreviations: AA, arch artery; CHD, congenital heart disease; CHTD, congenital hypo-thyroidism caused by thyroid dysgenesis; CIA, collagen-induced arthritis; CNV, copy num-ber variant; 3D, three dimensional; DAPI, 4�,6�-diamino-2-phenylindole; DGS, DiGeorgesyndrome; DIG, digoxigenin; EGFP, enhanced GFP; FISH, fluorescent WISH; GFP, greenfluorescent protein; HA, hypobranchial artery; hpf, hours postfertilization; MO, morpho-lino antisense oligonucleotide; NI, noninjected; NTN1, netrin-1; OFT, outflow tract; PDA,patent ductus arteriosus; PFA, phosphate-buffered paraformaldehyde; sb-MO, splice-blocking MO; SNP, single-nucleotide polymorphism; SYT17, synaptotagmin-17; tb-MO,translation-blocking MO; VSD, ventricular septal defect; WIF, whole-mount immunoflu-orescence; WISH, whole-mount in situ hybridization.

T H Y R O I D - T R H - T S H

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being less frequent. Congenital hypothyroidism caused bythyroid dysgenesis exists in the familial (2%) and sporadic(98%) forms (3), with a discordance rate of 92% betweenmonozygotic twins (4) and a female predominance (2);congenital heart disease is also predominantly sporadic (5)with a discordance rate of 90% between monochorionictwins (6).

Germline mutations in thyroid-related transcriptionfactors NKX2.1, FOXE1, and PAX8 have been identifiedin 3% of patients with sporadic CHTD and no cardiacdefects (7, 8). For NKX2.1 and PAX8, all mutations re-ported to date were monoallelic, and patients presentedwith orthotopic thyroid gland hypoplasia. Conversely,FOXE1 mutations have been found in the biallelic state inpatients presenting with athyreosis, cleft palate, and spikyhair (7). The lack of linkage to these genes in some mul-tiplex families with CHTD points to considerable geneticheterogeneity in this disorder (9). Two other genes,NKX2.5 and HHEX, have been implicated in thyroid andcardiac phenotypes based on human and genetic mousemodels (10–12). In transcriptome analysis of ectopic thy-roids, none of these transcription factors exhibited de-creased expression (13). Altogether these findings under-line the importance of identifying new genes associatedwith CHTD and congenital heart disease (CHD) by con-sidering the following: 1) sporadic de novo germline ge-netic events [ie, either de novo copy number variants(CNVs) or de novo point mutations], 2) the possibility ofmultiple hits (de novo or inherited) in modifier genes, 3)the possibility of low-penetrance variants, and 4) somaticepigenetic or genetic events (14).

CNVs have been recognized as a major source of ge-netic variability (15, 16) and have been shown to confersusceptibility to sporadic diseases (15, 17) such as autism(17) and CHD (18). An association between CHTD andchromosomal variants was demonstrated in two previousstudies performed with lower resolution than that used inthe present work (19, 20).

Like CHTD, CHD is a sporadic condition (5). An 8%co-occurrence of CHD in CHTD is greater than expectedby chance alone, given a global prevalence of CHD of0.6%–1% (21, 22). Therefore, CHD and CHTD mightshare common genetic or epigenetic etiologies. A link be-tween cardiovascular and thyroid development has pre-viously been demonstrated in mouse and zebrafish studiesrevealing coordinated morphogenetic processes (23) aswell as the occurrence of thyroid anomalies in mice andzebrafish models with defective cardiovascular develop-ment (23, 24). Therefore, we performed a pilot study byselecting patients with co-occurring CHD and CHTD tolook for shared genetic factors.

Subjects and Methods

Ethics statementThis study was approved by the Sainte Justine Ethics

Committee (ERB number 94). All of the parents providedwritten informed consent.

SubjectsTo determine whether rare CNVs are associated with

syndromic cases of CHTD, we selected patients withCHTD and CHD. First, the Zoom Endo database (Endo-crinology Service, Centre Hospitalier Universitaire Sainte-Justine) containing 158 patients with CHTD (diagnosisestablished by 99Tc scintigraphy) was merged with a car-diac echocardiography database containing echographiesof 20 000 different patients from the same institution.Nine patients were found to overlap, representing a prev-alence of 5.5% (9 of 158) for CHD among patients withCHTD, a prevalence consistent with an earlier survey byour group (2). To enrich for a rare phenotype likely to begenetically simpler, we selected three patients with a ven-tricular septal defect (VSD) for molecular studies. Theother six patients, with transient patent ductus arteriosus(PDA) and transient collagen-induced arthritis (CIA) typeII (CIA II), were excluded because transient PDA and CIAII are benign and observed in high proportion of otherwisenormal newborns; one case (patient 3) also had develop-mental delay and arthrogryposis (Figure 1). Clinical char-acteristics for each patient are reported in Table 1. Thecontrol cohort consisted of 203 ethnically matched indi-viduals with no evidence of thyroid or heart disease aftera medical history review, physical examination, electro-cardiogram, and echocardiography. Informed consentwas obtained from all participants. Cardiac and endocri-nological phenotyping of patients are reported in Table 1,and complete clinical case reports are available in supple-mental data.

Direct sequencingBlood was obtained from patients and parents using

peripheral venipuncture. DNA was extracted using apureLink genomic DNA minikit (Life Technologies). Ex-ons and intron-exon junctions were sequenced forNKX2.5, HHEX, NKX2.1, and PAX8. The single exon,including the polymorphic region encoding the alaninestretch, was sequenced for FOXE1. Primers and amplifi-cation conditions are available upon request.

Post hoc whole-exome sequencingFor the three patients, exome sequencing was per-

formed subsequently at the McGill University and Ge-nome Québec Innovation Center using the Agilent Sure-

2 Opitz et al Netrin-1 in Congenital Thyroid and Cardiac Defects Endocrinology

Select oligo capture library and Illumina HiSeq 2 � 100paired end reads. Details for exome sequencing and vari-ant analysis were performed as described in our previouspaper (25).

CNV detection analysisSamples were genotyped on the Affymetrix genome-

wide single-nucleotide polymorphism (SNP) Array 6.0 ac-cording to the manufacturer’s specifications. To increasespecificity, we used a merge procedure of two differentalgorithms (ie, GTC 3.0.2 from Affymetrix and Birdsuite

1.5.5 from the Broad Institute (Cambridge, Massachu-setts) to call CNVs, as published previously by our group(18) and as further described in the Supplemental Data.

Quantitative PCR validationCNVs found by genome-wide SNP array were vali-

dated using TaqMan gene copy number assays (AppliedBiosystems). Probes were designed using publicly avail-able software (http://www5.appliedbiosystems.com/tools/cnv/). The TaqMan assay identifications are listed in

Figure 1. Clinical pictures of patient 3 with CHD, thyroid ectopy, and arthrogryposis. Presenting patient features are shown; these include down-slanting palpebral fissures, high nasal bridge, hook-shaped nose, small mouth, normal palate, low-set ears with folded helices, and distalarthrogryposis involving fingers and toes (panels A–C). D, For the same patient, the ectopic thyroid gland is revealed by 99mTc sodiumpertechnetate scintigraphy.

Table 1. Phenotypic and genotypic characterization of the three patients

Case SexThyroidal

PhenotypeCardiac

PhenotypeOther

PhenotypeDirect Sequencing

NKX2.5a

ValidationTaq Man AssayIdentificationCopy Loss/Gain Chromosome Start End Size, kb Genes Classification

1 F Ectopy VSD, ASD ø R25C 0c – – – – – – – – –

2 M Athyreosis VSD ø wt 1 Loss 16p12.3 19 140 333 19 216 014 76 SYT17 Rare Inherited Confirmed by qPCR Hs01544221_cn

3 M Ectopy VSD, ASD, PDA Arthrogryposis wt 1 Loss 17p13.1 8 901 608 8 913 177 12 NTN1 Rare de Novo Confirmed by qPCR Hs05487409_cn

1 Loss 22q11.21 19 046 924 19 902 202 855 CRKL Rare de Novo Confirmed by qPCR Hs04079552_cn

2 Gain Y Karyotype 47XYY

Abbreviations: ASD, atrial septal defect; F, female; M, male; PDA, patent ductus arteriosus; qPCR, quantitative PCR; VSD, ventricular septal defect;wt, wild type.a Only positive results for NKX2.5 are presented. No mutations or rare variants were found in NKX2.1, FOXE2, PAX8, or HHEX.b Copy number, chromosome location with the start, end, and length of the rare CNVs with their classification, and major encompassing gene(University of California, Santa Clara, genome browser; HG18 assembly).c No rare deletion or duplication was validated in patient 1.

doi: 10.1210/en.2014-1628 endo.endojournals.org 3

Table 1 and a detailed protocol is provided in the Supple-mental Data.

Zebrafish embryo cultureZebrafish (Danio rerio) embryos were raised at 28.5°C

and staged in hours postfertilization (hpf) as described(26). Transgenic zebrafish lines tg(tg:mCherry) (23) andtg(kdrl:EGFP) (27) were used in this study. Embryos wereanesthetized in 0.016% tricaine (Sigma), fixed in 4%phosphate-buffered paraformaldehyde (PFA; Sigma)overnight at 4°C, washed in PBS containing 0.1% Tween20, gradually transferred to 100% methanol, and stored at�20°C until used for in situ hybridization or immunoflu-orescence analyses. All zebrafish work at the Institute ofInterdisciplinary Research in Molecular Human Biologyfollowed protocols approved by the Institutional AnimalCare and Use Committee.

Morpholino injectionsFor inhibition of ntn1a and ntn1b function, zebrafish

embryos were injected with morpholino antisense oligo-nucleotides (MOs) that have previously been validated fortheir knockdown specificity and efficacy (28–31). Toknock down the ntn1a function, 5–6 ng of a splice-block-ing MO (sb-MO; 5�-ATGATGGACTTACCGACA-CATTCGT-3�) were injected as previously described (28–30). To inhibit the ntn1b function, 4–6 ng of a translation-blocking MO (tb-MO; 5�-CGCACGTTACCAAAATCCTTATCAT-3�) wereinjected as previously described (28, 31). The standardcontrol MO designed by Gene Tools had the followingsequence: 5�-CCTCTTACCTCAGTTACAATTTATA-3�. Working solutions of MOs were prepared in 0.12 MKCl containing phenol red and 2–6 nL of MO solutionwas microinjected into the high yolk of one- to two-cellstage embryos. Inhibition of normal ntn1a mRNA splicingafter an sb-MO injection was verified as described (30)(Supplemental Figure 1).

Whole-mount in situ hybridization (WISH)DNA templates for synthesis of ntn1a, ntn1b, tg, and

nkx2.1a riboprobes were generated by PCR (see Supple-mental Table 1 for primer sequences). Plasmids for myl7and kdrl riboprobes have been used as described (32, 33).Single-color WISH was performed essentially as described(34). For dual-color WISH, riboprobes labeled withdigoxigenin (DIG) and dinitrophenol were used and se-quential alkaline phosphatase staining was performedwith BM Purple and Fast Red (Sigma) as described (23).Fluorescent WISH (FISH) using a DIG-labeled riboprobefor tg was performed as described (23). Antibodies used inWISH and FISH experiments are listed in Supplemental

Table 2. Stained embryos were postfixed in 4% PFA(Sigma) and embedded in 90% glycerol for whole-mountimaging or in 7% low melting point agarose (Lonza) forvibratome sectioning. Tissue sections at 50–60 �m thick-ness were cut on a Leica VT1000S vibratome and mountedin Glycergel (Dako). Images of stained sections were ac-quired using an Axiocam digital camera mounted on anAxioplan 2 microscope (Zeiss).

Whole-mount immunofluorescenceWhole-mount immunofluorescence (WIF) staining was

performed essentially as described (23). Specifications andsources of primary and secondary antibodies used to de-tect green fluorescent protein (GFP), mCherry, cardiactroponin T, and T4 in zebrafish embryos are provided inSupplemental Table 2. After WIF staining, specimens wereincubated in 4�,6�-diamino-2-phenylindole (DAPI) to la-bel cell nuclei and postfixed in 4% PFA. Combined FISHand WIF staining was performed as described (23) Con-focal images were acquired using an LSM 510 confocalmicroscope (Zeiss). Three-dimensional reconstruction ofconfocal stacks was performed using Zen 2010 D software(Zeiss).

Statistical analysesData sets from thyroid cell number measurements in

zebrafish were first analyzed for normal distribution (Kol-mogorov-Smirnov test) and homogeneity of variances(Bartlett’s test). If measurements of a response attribute, ora log-transformation of it, were found to be normally dis-tributed with equal variances, then an unpaired Student’st test was used for pair-wise comparison of stage-matchedexperimental groups. Statistical analyses were performedusing the software package GraphPad Prism 4.0 (Graph-Pad). Differences were considered significant at P � .05.

Results

Clinical ascertainmentBy merging data from our endocrinology and cardiol-

ogy clinical databases, we identified nine patients withcongenital hypothyroidism caused by thyroid dysgenesis(CHTD) and congenital heart defects (CHD). From thesenine, a subset of three patients was selected for molecularstudies, based on the clinical severity (ie, patients withventricular septum defects). Parental DNAs were avail-able for all three patients.

Direct sequencing of HHEX, NKX2.1, NKX2.5,FOXE1, and PAX8 and post hoc whole-exomesequencing

To exclude mutations in genes known to be associatedwith isolated CHTD (NKX2.1, FOXE1, and PAX8) or

with combined thyroid and cardiac defects (HHEX,NKX2.5), we sequenced these genes in all three patients.No candidate pathogenic mutations were detected inHHEX, NKX2.1, FOXE1, and PAX8. All patients werehomozygous for the FOXE1 14 alanine stretch polymor-phism (35). Subsequently, we performed whole-exome se-quencing as described elsewhere (25) and no additionalpathogenic variants were found in these patients.

Identification of a rare inherited heterozygousvariant of NKX2.5 in patient 1

In patient 1, we identified a c.73C�T transition inNKX2.5 (rs28936670, minor allele frequency (0.003), re-sulting in a pArg25Cys change. This variant was inheritedfrom the unaffected father (Table 1). No rare deletion orduplication was validated in that patient.

CNV detectionCNV detection in patients and their parents was per-

formed using the Affymetrix genome-wide SNP Array 6.0.All detected variants were not found in 203 ethnicallymatched controls. No rare CNV were found in patient 1.CNV found in patients 2 and 3 are described below.

Identification of a rare inherited deletion ofsynaptotagmin-17 (SYT17) in patient 2

In patient 2, we identified a deletion of 76 kb encom-passing SYT17 and the undefined locus UNQ5810 onchromosome 16p13.2 (Table 1). This variant was inher-ited from the unaffected father. SYT17 is ubiquitouslyexpressed, with high levels in the thyroid and heart.SYT17 belongs to the group of synaptotagmin-soluble N-ethylmaleimide sensitive fusion factor attachment proteinreceptor interactors, although its possible involvement inCTHD and CHD have not been examined to date.

Identification of two rare de novo CNVs and a 47,XYY karyotype in patient 3

In patient 3 (Figure 1), we identified two rare de novoCNVs as well as a 47, XYY karyotype (Table 1). The firstde novo deletion encompassed a total of 855 kb of se-quence on chromosome 22q11, corresponding to an atyp-ical 22q11 deletion syndrome (36). This interval contains33 genes that would thus be haploinsufficient, includingCRKL. Somewhat surprisingly, a routine FISH performedwith the usual TUPLE1 probe revealed no 22q11 deletion.However, FISH using BAC RP11–801020 confirmed thedeletion in the distal DiGeorge syndrome (DGS) region inthe index case, whereas parental FISH results were nor-mal. The second CNV was a de novo 12-kb deletion be-tween the second and third exons of netrin-1 (NTN1) onchromosome 17p13.1. NTN1 is a laminin-related se-

creted protein that acts as an axon guidance molecule dur-ing neural development (37).

Netrin1 mRNA expression in zebrafish embryosBecause Netrin1 is implicated in the regulation of var-

ious developmental processes including angiogenesis,nonneuronal cell migration, and epithelial morphogenesis(38, 39), we decided to use zebrafish embryos as a modelto characterize the expression and function of Netrin1with respect to thyroid and cardiovascular development.In zebrafish, two paralogous homologs of human NTN1are expressed, ntn1a (40) and ntn1b (41). Zebrafish ntn1aand ntn1b act as axon guidance molecules, and ntn1a hasalso been implicated in vascular development (30, 42). Wefirst used WISH to examine spatiotemporal patterns ofntn1a and ntn1b expression in the thyroid/pharyngeal re-gion for which detailed expression data were not yet avail-able. For this purpose, embryos and larvae were fixed atvarious developmental stages throughout their develop-ment between 24 and 100 hpf. Thyroid specification inzebrafish occurs around 24 hpf (43) and the thyroid pri-mordium can be stained by WISH using a nkx2.1a ribo-probe (Figure 2A). For ntn1a, we did not detect any no-table expression in the thyroid/pharyngeal region ofzebrafish embryos at 24, 26, 30, and 34 hpf (Figure 2B anddata not shown). Robust ntn1a expression became detect-able in the pharyngeal region from 36 hpf onwards (Figure2, C–E). Dual-color WISH revealed pharyngeal ntn1a ex-pression domains rostral and lateral to the thyroid pri-mordium, but ntn1a was not expressed in the thyroid pri-mordium itself (Figure 2, F–H). Instead, ntn1a wasexpressed in the pharyngeal mesenchyme surrounding theaortic arch arteries (Figure 2, I and J). For ntn1b, we de-tected, if any, only a very weak staining of the pharyngealregion at 24, 26, 30, 34, 36, 38, 46, 48, 55, and 60 hpf(Figure 2, K–N, and data not shown). Robust ntn1b ex-pression became detectable at 72 hpf in lateral pharyngealregions (Figure 2O). Dual-color WISH showed no detect-able ntn1b expression in thyroid cells at any stage exam-ined (Figure 2, P–R, and data not shown), and vibratomesections of stained embryos revealed no ntn1b mRNA ex-pression in the pharyngeal arch region (Figure 2S). How-ever, between 46 and 55 hpf, the thyroid primordium wastransiently apposed to ntn1b-expressing cardiac tissue(Figure 2, N and T, and data not shown).

ntn1a knockdown causes defective cardiovascularand thyroid development

Although ntn1a-morphants have been reported for ner-vous system and vascular development (28, 30, 31), nodata have been available concerning thyroid developmentin ntn1a-deficient embryos. To test whether ntn1a is re-

quired for normal thyroid development, we knockeddown ntn1a function in zebrafish embryos using a previ-ously validated ntn1a sb-MO (28–30). Injection of 5–6 ngntn1a sb-MO efficiently prevented normal ntn1a mRNAsplicing and closely recapitulated previously described ef-fects patterns on the trunk vasculature (Supplemental Fig-ure 1). All results reported below have been obtained usingthis MO concentration.

To examine early thyroid and cardiac development inntn1a-deficient embryos, we performed dual-color WISHfor cardiac and thyroid markers in 28- and 55-hpf em-bryos. WISH staining of embryos with the myocardium-specific myl7 probe revealed cardiac laterality defects inntn1a-morphants. The first bilateral symmetry breakingevent during zebrafish heart development is a leftwarddisplacement of the cardiac cone, a process called cardiacjogging, which results in a leftward positioning of the ve-nous pole relative to the midline. The direction of cardiacjogging is regulated by left-right signaling (44), and a nor-mal leftward positioning of the venous pole (left jogging)was observed in noninjected (NI) embryos (NI-controls)

and embryos injected with control-MO (MO-controls) at28 hpf (Figure 3, A and B). In contrast, the direction ofcardiac jogging was randomized in ntn1a-morphants (Fig-ure 3U) with 42%, 28%, and 30% of ntn1a-morphantsdisplaying left heart jogging (Figure 3C), a no-jog pheno-type (Figure 3D), and right heart jogging (Figure 3E), re-spectively. A second important event involved in estab-lishing laterality of the zebrafish heart is the process ofcardiac looping occurring between 36 and 48 hpf (44).When examined at 55 hpf, hearts of NI-controls (Figure3F) and MO-controls (Figure 3G) showed correct D-loop-ing (ventricle positioned right to the atrium), whereasntn1a-morphants displayed abnormal heart looping (Fig-ure 3V). Only 47% of ntn1a-morphants showed D-loopedhearts (Figure 3H), 26% had unlooped midline hearts(Figure 3I) and 27% showed a reversed looping with theventricle positioned left to the atrium (Figure 3J).

Although cardiac jogging and subsequent cardiac loop-ing are discrete processes, the direction of cardiac joggingis generally considered a good predictor for cardiac loop-ing phenotypes (45). Our experiments were not designed

Figure 2. Expression of ntn1a mRNA and ntn1b mRNA during zebrafish development. A–E, During early thyroid development (from 24 to 36hpf), ntn1a mRNA is not expressed in the region of the thyroid primordium (see thyroidal nkx2.1a expression domain marked by arrowhead inpanel A). Pharyngeal expression of ntn1a is detectable in whole-mount embryos from 36 to 72 hpf (see arrowheads in panels C–E). F–J, Vibratomesections of dual-color-stained embryos revealed that ntn1a expression in the pharyngeal region does not include the thyroid primordium (markedby nkx2.1a staining, arrowhead in panels F–H). Parasagittal sections (see panels I and J) show strong ntn1a expression in the pharyngealmesenchyme (arrows in panel I) surrounding the aortic arch arteries (marked by kdrl staining and indicated as numbers 1, 3, 4, 5, and 6 in panel J).Sagittal (panels F and G), transverse (panel H), and parasagittal sections (panels I and J) are shown. K–O, ntn1b is very weakly expressed in thepharyngeal region between 24 and 60 hpf (panels K–M). From 46 to 60 hpf, a transient weak ntn1b expression was detected in the zebrafishheart (asterisk in panel N). At later stages, ntn1b is expressed in lateral pharyngeal regions (arrowheads in panel O). P–T, Vibratome sections ofdual-color-stained embryos revealed that ntn1b expression is absent in the thyroid primordium (marked by nkx2.1a staining, arrowheads in panelsP–R) at all stages examined. Very weak, diffuse ntn1b staining was present in parasagittal sections (see panel S) at the level of the pharyngealarches. Sagittal sections (see panel T) show that thyroid cells are near ntn1b-expressing cardiac tissue between 46 and 55 hpf. Sagittal (panels P,Q, and T), transverse (panel R), and parasagittal sections (panel S) are shown. Scale bar, 100 �m (A–E and (K–O); 50 �m (F–J and P–T).

to address specifically the relationship between joggingdirectionality and subsequent cardiac looping for individ-ual embryos, but the observed frequencies of jogging andlooping anomalies in ntn1a-morphants are consistentwith a model in which left jogging (42%) is expected to beaccompanied by D looping (47%), right jogging (30%) byreversed looping (27%), and a no-jog phenotype (28%)would result in unlooped midline hearts (26%).

WISH staining of the thyroid marker nkx2.1a in 28 hpf

embryos did not reveal gross differences in size, shape, andlocation of the thyroid primordium between NI-controls(n � 47), MO-controls (n � 104), and ntn1a-morphants(n � 116) (Figure 3, A–E). At 55 hpf, however, 48% ofntn1a-morphants (n � 46 of 95) presented an aberrantthyroid morphology. In contrast to the compact midlinethyroids of NI-controls (n � 43 of 45) and MO-controls(n � 83 of 86), thyroid tissue of ntn1a-morphants were notlimited to the midline and showed irregular lateral expan-

Figure 3. ntn1a-deficient embryos display defects in thyroid and cardiac development. A–D, Dual-color WISH of nkx2.1a and myl7 expression inNI embryos, embryos injected with control morpholino (co-MO), and embryos injected with ntn1a MO (ntn1a-MO). myl7 staining of the heart tuberevealed normal leftward positioning of the venous pole (left jog) in NI and co-MO embryos, whereas heart jogging was randomized in ntn1a-morphants (panels C–E). Thyroidal nkx2.1a expression (arrowhead) was not different between experimental groups at 28 hpf. Dorsal views areshown, anterior is to the top. F–O, Dual-color WISH of tg and myl7 expression in 55-hpf embryos (frontal view). myl7 staining showed correctheart looping (D loop) with the ventricle (V) positioned right to the atrium (A) in NI and co-MO embryos (see panels F and G). ntn1a-morphantsdisplayed randomization of heart looping (see panels H–J) including midline hearts and hearts with reversed looping (L loop). In addition, ntn1a-morphants have irregularly shaped thyroid primordia (arrowheads in panels F–J). In contrast to the compact and slightly ovoid thyroid primordiumof control embryos (see panels K and L), ntn1a-morphants displayed unilaterally (see panels M and O) or bilaterally (see panels N) expanded thyroidtissue. Magnified views of the thyroid region in panels K and L are not necessarily from the same embryos as shown in panels F–J. P–T, WISHanalysis of tg expression at 80 hpf showed a normal progressive expansion of thyroid tissue along the anterior-posterior axis in control embryos(see panels P and Q). In ntn1a-morphants, thyroid tissue was disorganized and often mislocated away from the midline (see panels R, S, and T).Ventral views are shown, anterior is to the top. U and V, Quantification of the number of embryos displaying defects of heart tube jogging at 28hpf (panel V) and heart looping at 55 hpf (panel U) as determined by myl7 staining. Results are presented as the percentage of embryos displayinga particular phenotype, and N denotes the total number of specimen analyzed for each treatment group. Scale bar, 100 �m (A–J), 50 �m (K–T).

sions (Figure 3, M–O). Laterally expanding thyroid tissuewas observed at similar frequencies on the left (n � 17 of95) and the right side (n � 19 of 95); bilateral thyroidexpansions were less frequent (n � 10 of 95). At approx-imately 55–60 hpf, the thyroid starts to expand along thepharyngeal midline and tg staining of 80 hpf control em-bryos showed the progressive anterior-posterior (AP) ex-pansion of thyroid tissue (Figure 3, P and Q). In mostntn1a-morphants (n � 52 of 80), however, this AP ex-pansion was defective, and ntn1a-morphants displayedirregularly positioned clusters of thyroid cells close to theheart outflow tract (OFT) (Figure 3, R and S) as well aslaterally misplaced thyroid tissue (Figure 3T). No thyroidor cardiac laterality defects were detected in embryos in-jected with a tb-MO targeting ntn1b (data not shown).

The irregular thyroid morphologies of ntn1a-mor-phants resembled thyroid phenotypes previously observedin zebrafish embryos with defects in pharyngeal vascula-ture morphogenesis (23). Using transgenic tg(kdrl:EGFP)embryos expressing enhanced green fluorescent protein(EGFP) in endothelial cells, we detected gross malforma-tions of the pharyngeal vasculature after injection of ntn1asb-MO but not control-MO or ntn1b tb-MO (Figure 4,A–C). Similar to mammalian embryos, the zebrafish aorticarch artery (AA) network consists of paired bilateral ar-teries that connect the heart OFT to the dorsal aortae.Confocal microscopy of 55 hpf tg(kdrl:EGFP) embryosand three-dimensional (3D) reconstruction of the pharyn-geal vasculature revealed a spectrum of defects in AA mor-phogenesis in 63% of ntn1a-morphants (Figure 4, D–H).Although the AA1 and the branchial AAs 3–6 were clearlyformed in 55-hpf control embryos, perturbed AA mor-phogenesis in ntn1a-morphants ranged from AA hypopla-sia to severe underdevelopment of the entire AA system(Figure 4, K–M). AA malformations in ntn1a-morphantswere further accompanied by hypobranchial artery (HA)dysplasia and failure to form a paired ventral aorta con-necting each AA to the heart OFT.

Confocal microscopy of double transgenic tg(tg:mCherry;kdrl:EGFP) embryos, expressing mCherry inthyroid and EGFP in endothelial cells, showed that aber-rant lateral thyroid expansion occurred predominantly inntn1a-morphants displaying abnormal HA morphologies(Figure 4, I–M, and Figure 5). Confocal analyses of 80-hpfembryos showed that lack of a normal AP expansion ofthyroid tissue in ntn1a-morphants was correlated with anoverall poorly developed hypobranchial vasculature in-cluding the defective AA and HA formation (Figure 4,N–R). When counting the number of thyroid cells (cellsdouble positive for mCherry and DAPI) in 80 hpf tg(tg:mCherry) embryos, no differences were detected betweenMO-controls and ntn1a-morphants (Figure 6). At 100

hpf, however, thyroid tissue of ntn1a-morphants con-tained significantly fewer thyroid cells than MO-controls(Figure 6). On the contrary, the functional maturation ofthyroid tissue appeared largely unaffected in ntn1a-mor-phants as judged by their capacity to form functional fol-licles producing the thyroid hormone T4 (Figure 4, S–V).

Discussion

The etiology of CHTD is one of the remaining enigmas inthe pathophysiology of thyroid diseases (46). CHTD is asporadic condition that has a discordance rate of 92%between monozygotic twins (4) and a significant associ-ation with congenital heart defects (2, 47). This promptedus to consider de novo germline genetic events (ie, de novoCNVs and/or point mutations) as the underlying cause. Inthis pilot study, we used a combination of targeted can-didate gene sequencing and high-density CNV analysis toassess the role of rare alleles with major effects in CHD andCHTD. We found rare variants in all three patients, andwe selected netrin-1 as the biologically most plausible con-tributory factor for functional studies in zebrafish. Thisstudy also underlines the value of combining phenotypeand genotype profiling with zebrafish functional studies touncover new pathogenic genes from a small cohort of pa-tients (11, 48).

In patient 3 we found three potentially relevant rarestructural variants: an 47, XYY karyotype; an atypical22q11 deletion; and an 17q deletion.

First, standard karyotyping revealed a 47, XYY chro-mosome constitution that was corroborated by high-res-olution karyotyping. An association between congenitalhypothyroidism and sex chromosome aneuploidy (ie, es-pecially in 47, XXY) has been previously reported (49).Thorwarth et al (20) also reported a wide Y duplication ina patient with thyroid hypoplasia and a VSD.

The 22q11 deletion in patient 3 overlaps with deletionsassociated with DGS in which thyroid dysfunction is avariable component of the phenotype. However, geno-type-phenotype correlations have been restricted to classicdeletions as defined by the TUPLE1 probe (50). The TU-PLE1 standard probe did not detect the deletion in ourpatient even though the clinical picture was typical forDGS. According to studies in mice, deletion of Tbx1 fullyreplicates the thyroid phenotype seen in DGS (51), yet thisgene was not deleted in patient 3. A role for Crkl in an-teroposterior patterning of the pharyngeal apparatus hasbeen described; this process requires the cooperation withTbx1 and local retinoic acid signaling (52). Of note, evenif up to 50% of classical DGS present with thyroid hyp-oplasia (53), thyroid ectopy is not reported in DGS, and in

a series of atypical DGS (ie, CRKL deletion), no patientswere reported to have hypothyroidism (36). Therefore,CRKL deletion is associated with neither thyroid ectopynor hypothyroidism, and we did not select CRKL for fur-ther validation with functional assays.

More importantly, patient 3 carries a de novo 12-kbdeletion on chromosome 17p13.1 within the NTN1 gene,

which is predicted pathogenic because two consecutiveexons are deleted. NTN1 is of great interest in light offindings that place this gene in the Sonic hedgehog signal-ing pathway and implicate it as a physical and functionalinteractor of the Down syndrome cell adhesion molecule,a gene implicated in the cardiac phenotype of Down syn-drome patients (54–56). NTN1 is crucial for normal brain

Figure 4. Defective morphogenesis of pharyngeal vessels is associated with aberrant localization of thyroid tissue in ntn1a-deficient embryos. A–C, GFP immunofluorescence staining of 80-hpf transgenic tg(kdrl:EGFP) embryos injected with co-MO, ntn1a-MO, or ntn1b-MO. Pharyngealvascular development was defective in ntn1a-deficient embryos (see panel B) but not in embryos injected with ntn1b-MO (see panel C). Note thatdefective formation of AAs and associated vessels (red arrows in panels A–C) occurred despite an overall normal vascular development in ntn1a-morphants. 3D reconstructions of confocal images are shown, lateral view, anterior is to the left. D–H, GFP immunofluorescence staining of 55-hpftransgenic tg(kdrl:EGFP) embryos. NI embryos and co-MO-injected embryos had formed bilateral pairs of five AAs (indicated as numbers 1, 3, 4, 5,and 6). Formation of the HA is evident, and a paired ventral aorta (VA) has formed connecting the heart outflow tract via branchial AAs 3–6 to thelateral dorsal aorta. In contrast, ntn1a-morphants displayed defective pharyngeal vessel morphogenesis, ranging from limited and incomplete AAformation to an almost complete absence of branchial AAs (see panels F–H). Defects in VA and HA formation correlated with the severity of AAabnormalities in ntn1a-morphants (see panels F–H). 3D reconstructions of confocal images are shown, lateral view, anterior is to the left. I–R,Confocal microscopy of transgenic tg(tg:mCherry; kdrl:EGFP) embryos after immunofluorescence staining for mCherry (thyroid, red), GFP(endothelium/endocardium, green), and cardiac troponin T (myocardium, white). Thyroid anomalies including ectopic lateral thyroid expansionwere exclusively found in ntn1a-morphants displaying defects in pharyngeal vascular development, particularly defects in HA morphogenesis. 3Dreconstructions of confocal images are shown, ventral view, anterior is to the left. S–V, Despite defects in size, shape, and position of thyroidtissue, follicle formation and functional maturation of thyroid tissue appeared unaffected in ntn1a-morphants. Panels S and T show confocalsections of embryos (ventral view, anterior to the left) after double staining for tg mRNA and colloidal T4. Panels U and V show confocalprojections of embryos (ventral view, anterior to the left) after colloidal T4 staining. Scale bar, 200 �m (A–C), 50 �m (D–R), 20 �m (S-V).

development but recent studies have implicated NTN1signaling also in the regulation of nonneuronal cell mi-gration and survival and vascular development as well asepithelial cell adhesion and migration during lung andpancreas morphogenesis (57, 58). Similar to lungs andpancreas, the thyroid is derived from foregut endoderm,but a role of netrin-1 action during thyroid developmenthas not yet been reported.

Although the clinical significance of the detected vari-ants at the NTN1 locus remains to be determined, ourfunctional studies in zebrafish embryos provide significantevidence linking netrin-1 action with pharyngeal vesseland thyroid morphogenesis. First, we detected dynamicexpression of zebrafish homologs of NTN1 in the pha-ryngeal region, although neither ntn1a or ntn1b was ex-pressed in the developing thyroid itself. Instead, ntn1a wasexpressed in pharyngeal tissue surrounding the developingAAs, and defective AA formation was one hallmark of thentn1a loss-of-functionphenotype.Althoughdefective thy-roid morphogenesis became apparent at 55 hpf, after thy-roid separation from the pharyngeal floor, perturbed AAformation was already evident at earlier stages. Anotherkey observation was that the presence of irregularlyshaped and ectopically located thyroid tissue was strictlyassociated with aberrant morphogenesis of pharyngealvessels (eg, the HA) that are important for guiding latethyroid relocalization (23). Together these observationssuggest that abnormal pharyngeal vessel formation mightbe the primary effect of ntn1a deficiency, whereas the thy-roid anomalies most likely represent a secondary responseto the lack of a proper guidance function exerted by dys-plastic pharyngeal vessels. In this regard, our data rein-force the concept that embryonic blood vessels play a crit-ical role in thyroid organogenesis (24, 59) and thatvascular anomalies may account for certain cases of

CHTD. Consistently, there are clinical observations ofvascular malformations (eg, hypoplasia or agenesis of thy-roid arteries) in some cases of ectopic thyroids in humans(60, 61). Ntn1a-deficient zebrafish embryos also dis-played cardiac laterality defects, but neither abnormalheart jogging nor heart looping was correlated with thepresence of thyroid anomalies.

Two other inherited variants were found. First, theNKX2.5 p.R25C variant identified in patient 1 was pre-viously found in patients with both CHD and CHTD (11).In addition, functional assays suggested that the R25Cmutant exhibits impaired binding and transactivationproperties (11, 62). Because this variant was also found inthe father of patient 1 as well as at low frequency in somecontrol series with a minor allele frequency of 0.003 ondbSNP, we postulate that it constitutes a reduced pen-etrance risk allele. Second, we found a deletion in SYT17in patient 2, again inherited from an unaffected father.Given that SYT17 deletion was inherited from the healthyfather, we did not select this gene for further validationwith functional assays (48).

Our study implicates known pathways of thyroid andheart development and replicates previous results suggest-ing a possible contributory role for the NKX2.5 p.R25Cvariant. The incomplete penetrance of this variant in ourpatient 1 is compatible with either genetic or environmen-tal modifiers. These observations suggest a complex modeof inheritance in CHTD and CHD, which are now genet-ically traceable using modern high-resolution platforms(63).

Based on the high yield of rare variants identified in thispilot analysis, future studies in patients with CHTD andCHD are warranted, and these studies should include acomprehensive analysis of protein-coding mutations andstructural genomic variation. High-resolution chip plat-

Figure 5. Three-dimensional reconstruction of confocal images of the heart OFT region of 55 hpf tg(tg:mCherry;kdrl:EGFP) embryos expressingmCherry (red) in thyroid cells and EGFP (green) in endothelial cells and endocardium. A, In embryos injected with a co-MO, the endothelial cells ofthe HA embrace the thyroid primordium, which is present as a compact ovoid midline structure located rostral to the heart OFT. B and C, Incontrast, embryos injected with a ntn1a-MO displayed irregular thyroid morphologies. Uni- or biaterally expanding thyroid tissue waspredominantly observed along the course of an abnormally bifurcated HA. AA1, aortic arch artery 1. Scale, 20 �m.

forms and whole genome sequencing in trios with affectedCHTD/CHD children will yield insight into complex in-heritance patterns affecting interacting pathways of em-bryonic development of the thyroid and heart.

Acknowledgments

We thank the patients and their parents for their cooperation. Wealso thank Drs C. Deal and G. Van Vliet (Centre HospitalierUniversitaire Sainte-Justine, University of Montréal) for theircontinuous support during this project. In addition, we thankFabien Magne for technical assistance. We also thank C. Stacher-Hörndli (University of Utah Medical Center, Salt Lake City,Utah) and G. Peng (Fudan University, Shanghai, China) for theirgenerosity with morpholino antisense oligonucleotide reagents,V. Janssens (Université Libre de Bruxelles, Brussels, Belgium) fortechnical assistance in the morpholinos injections, J.-M. Vander-winden, and F. Bollet-Quivogne (Light Microscopy Facility, Uni-versité Libre de Bruxelles) for technical assistance in the confocalmicroscopy.

Address all correspondence and requests for reprints to:Johnny Deladoëy, MD, PhD, Centre Hospitalier UniversitaireSainte-Justine, 3175 Côte Sainte-Catherine, Montréal Québec,Canada H3T 1C5. E-mail: johnny.deladoey@umontreal.ca; orGregor Andelfinger, MD, PhD, Centre Hospitalier UniversitaireSainte-Justine, 3175 Côte Sainte-Catherine, Montréal, Québec,Canada H3T 1C5. E-mail: gregor.andelfinger@umontreal.ca; orSabine Costagliola, Institute of Interdisciplinary Research inMolecular Human Biology, Université Libre de Bruxelles, Routede Lennik 808, 1070 Brussels, Belgium. E-mail:scostag@ulb.ac.be.

This work was supported by grants from the Canadian In-stitutes of Health Research (to J.D. and G.A.); by the Fonds deRecherche du Québec-Santé (to J.D. and G.A.); by GrantGMHD79045 from the Heart and Stroke Foundation of Canada(to G.A.); by the Girafonds/Fondation du Centre HospitalierUniversitaire Sainte-Justine (to J.D.); by the European Society forPediatric Endocrinology (ESPE Research Unit grant, to J.D. andS.C.); by Grant FRSM 3_4598_12 from the Belgian Fonds de laRecherche Scientifique Medicale (to S.C.); by Grant ARCAUWB-2012–12/17-ULB3 from the Action de Recherche Con-certée de la Communauté Française de Belgique (to S.C.); by theFonds d’Encouragement à la Recherche; and grants from theBelgian National Fund for Scientific Research (FNRS). R.O. is anFNRS Postdoctoral Researcher, I.V. is an FNRS Research Fel-low, A.T. is an FNRS Short-Term Foreign Postdoctoral Fellow,and S.C. is an FNRS Senior Research Associate.

Disclosure Summary: The authors have nothing to disclose.

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