Transient PAX8 Expression in Islets During PregnancyCorrelates With b-Cell Survival, Revealing a NovelCandidate Gene in Gestational Diabetes MellitusAlejandro Martin-Montalvo,1 Livia López-Noriega,1 Carmen Jiménez-Moreno,1 Amanda Herranz,2

Petra I. Lorenzo,1 Nadia Cobo-Vuilleumier,1 Alejandra Tamayo,2 Cristian González-Guerrero,2

Jonathan S.W.R. Hofsteede,2 Fanny Lebreton,3 Domenico Bosco,3 Miguel García Toscano,4 Lucrecia Herranz,2

Joao Anselmo,5 José Carlos Moreno,2 and Benoit R. Gauthier1

Diabetes 2019;68:109–118 | https://doi.org/10.2337/db18-0285

Transient Pax8 expression was reported in mouse isletsduring gestation, whereas a genome-wide linkage andadmixture mapping study highlighted PAX8 as a candi-date gene for diabetes mellitus (DM). We sought thesignificance of PAX8 expression in mouse and humanislet biology.PAX8was induced in gestatingmouse isletsand in human islets treated with recombinant prolactin.Global gene expression profiling of human and mouseislets overexpressing the corresponding species-specific PAX8 revealed the modulation of distinct geneticpathways that converge on cell survival. Accordingly,apoptosis was reduced in PAX8-overexpressing islets.These findings support that PAX8 could be a candidategene for the study of gestational DM (GDM). PAX8 wasgenotyped in patients with GDM and gestational thyroiddysfunction (GTD), a pathology commonly found inpatients with mutations on PAX8. A novel missensePAX8 mutation (p.T356M, c.1067C>T) was identified ina female diagnosed with GDM and GTD as well as in herfather with type 2 DM but was absent in control patients.The p.T356M variant did not alter protein stability orcellular localization, whereas its transactivation activitywas hindered. In parallel, a retrospective clinical analysisuncovered that a pregnant female harboring a secondPAX8 mutation (p.P25R, c.74C>G) previously reported tocause congenital hypothyroidism also developed GDM.

These data indicate that increased expression of PAX8affects islet viability and that PAX8 could be consideredas a candidate gene for the study of GDM.

During gestation, maternal metabolic adaptations are es-sential to ensure the health of the mother and the viabilityof the fetus. One of the organs that must undergo a pro-found adaptation during pregnancy is the endocrinepancreas. The islet b-cell mass expands as an adaptiveresponse to the progressive insulin resistance that devel-ops in the pregnant female to favor nutrient accessibilityto the fetus (1). This mechanism of b-cell mass expansionincludes b-cell hypertrophy, increased b-cell proliferation,and increased prosurvival/antiapoptotic signaling (2). Fail-ure to adapt may result in the development of gestationaldiabetes mellitus (GDM), with concomitant health issuesnot only for the mother but also for the fetus. The under-lying molecular mechanisms triggering GDM remainlargely uncharted (3). Toward identifying these molecularpathways, two independent studies have analyzed thetranscriptome landscape of islets isolated from gestatingmice (2,4). Of note, one of the most upregulated geneswas the transcription factor paired box 8 (PAX8) (2).Historically, PAX8 is known for its essential role in the

1Pancreatic Islet Development and Regeneration Unit/Laboratory of Aging Biology,Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER,Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Seville, Spain2Thyroid Molecular Laboratory, Institute for Medical and Molecular Genetics, LaPaz University Hospital, Autonomous University of Madrid, Madrid, Spain3Cell Isolation and Transplantation Center, Department of Surgery, GenevaUniversity Hospitals, Geneva, Switzerland4Amarna Therapeutics, Seville, Spain5Department of Endocrinology and Nutrition, Hospital Divino Espírito Santo, PontaDelgada, Portugal

Corresponding author: Benoit R. Gauthier, benoit.gauthier@cabimer.es;Alejandro Martin-Montalvo, alejandro.martinmontalvo@cabimer.es; or JoséCarlos Moreno, josecarlos.moreno@salud.madrid.org

Received 8 March 2018 and accepted 17 October 2018

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0285/-/DC1.

© 2018 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

Diabetes Volume 68, January 2019 109

ISLETSTUDIES

development and function of the thyroid and urogenitalsystem, maintaining its expression in adult kidney andthyroid gland (5). In agreement with this described patternof expression, mutations/polymorphisms in PAX8 havebeen associated with hypothyroidism and urorenal abnor-malities, whereas its overexpression correlates with tumorformation in these and other tissues (6,7).

We have previously reported that Pax8 is expressedneither during mouse pancreatic development nor in adultislets under normal physiological conditions (8,9). Thisfact raises the prospect that PAX8 expression likely willbe induced transiently under specific physiological con-ditions, such as pregnancy, which may confer benefits toislets. Consistent with this beneficial effect of PAX8 undersituations of increased insulin demand, a single genome-wide linkage and admixture mapping study has high-lighted PAX8 as a putative type 2 diabetes mellitus (T2DM)candidate gene in African American descendants (10). Inthe current study, we sought to validate PAX8 expressionand determine its physiological function during preg-nancy. We observed that PAX8 is transiently expressedin mouse under gestating conditions as well as in humanislets exposed to conditions mimicking gestation in vitro.Lentiviral-mediated PAX8 overexpression in islets modu-lates genetic pathways–implicated immunomodulation,protein processing, and cell survival. More importantly,we identified a yet undescribed missense mutation of PAX8(p.T356M, c.1067C.T) in a family pedigree in whicha female developed GDM while her father developedT2DM. Functional analysis demonstrated that the p.T356Mmutation significantly decreased PAX8 transactivation po-tential. In addition, a retrospective clinical analysis uncov-ered that GDM developed in a pregnant female of a familialpedigree that harbors a previously described PAX8-P25Rmutation associated with congenital hypothyroidism.

RESEARCH DESIGN AND METHODS

MiceC57BL/6J mice were purchased from JANVIER LABS(Saint-Berthevin, France). Five-month-old C57BL/6J preg-nant females were euthanized at post coitum day (pcd)10.5, 14.5, 16.5, and 18.5 for islet isolation. Pax8 knockoutmice were procured from the European Mouse MutantArchive network. Mouse experiments were approvedby the Centro Andaluz de Biología Molecular y MedicinaRegenerativa (CABIMER) animal committee and per-formed in accordance with Spanish law on animal use(RD 53/2013).

Oral Glucose Tolerance TestPregnant wild-type (Wt) and heterozygous Pax8 micewere subjected to an oral glucose tolerance test (OGTT)at pcd 14.5 as previously reported (11).

Pancreatic Islet Procurement and TreatmentMouse islets were isolated using the intraductal collage-nase perfusion protocol (12,13). Human islets were

procured from the Cell Isolation and Transplanta-tion Center (Geneva, Switzerland). Characteristics ofhuman islet preparations are presented in SupplementaryTable 1. Islets were cultured as previously described (14).Where indicated, murine islets were treated for 16 h withcytokines at 48 h after lentiviral infection (interferon-g[IFN-g 1,000 units/mL, interleukin-1b [IL-1b 100units/mL, and tumor necrosis factor-a [TNF-a 1,000units/mL). Where indicated, human islets were treatedwith recombinant human prolactin (200 ng/mL daily) forup to 96 h (Sigma-Aldrich, Madrid, Spain). Before treat-ments, human islets were precultured in media supple-mented with charcoal-treated serum for 2 days to removehormonal contaminants.

Quantitative RT-PCRTotal RNA was extracted using the RNeasy Micro Kit(QIAGEN). cDNA using 0.5–1 mg RNA was synthe-sized using SuperScript II Reverse Transcriptase (ThermoFisher Scientific, Madrid, Spain). The RT-PCR was per-formed on individual cDNAs using SYBR Green (RocheDiagnostics, Mannheim, Germany). Primer sequences arepresented in Supplementary Table 2. The mRNA expres-sion was calculated by the 22DDCT method and normalizedto the expression of cyclophilin, RPS29, and/or b-actin (12).

Generation of PAX8 Lentiviruses and TransductionThe human and murine PAX8Wt cDNAs (Origene, Rock-ville, MD) were subcloned into the pHRSIN DUAL-GFP easy vector provided by J.A. Pintor-Toro (CABIMER).To generate control lentiviruses for mock transductions,the empty vector was used. This vector allows the clon-ing and expression of a gene of interest under the controlof the spleen focus-forming virus promoter while theconstitutive ubiquitin promoter regulates expression ofthe reporter green fluorescent protein (GFP). Lentivirusamplification, purification, and transduction were per-formed as previously described (13). Islet transductionefficiency was estimated by flow cytometry (FACSCalibur;BD Biosciences, Madrid, Spain) using ;20 disaggregatedislets.

Islet Viability, Proliferation, and Insulin SecretionAssessmentApoptosis and proliferation were assessed using cell deathdetection and BrdU cell proliferation ELISA, respectively(catalog #11647229001; Sigma-Aldrich, Madrid, Spain).BrdU (10 mmol/L) was added during the last 24 h of theassay at each time point. Glucose-stimulated insulin secre-tion (GSIS) was performed as previously described (14).

ImmunohistochemistryIslet embedding and immunohistochemistry analysis wereperformed as previously described (12). A list of primaryand secondary antibodies is provided in SupplementaryTable 3. Images were acquired using a Leica microscope(AF6000; Leica, Milton Keynes, U.K.).

110 PAX8 and Pancreatic Islets in Pregnancy Diabetes Volume 68, January 2019

Transcriptome ProfilingcRNA preparations from human and murine islets werehybridized to GeneChip Human Gene 2.0 ST Array Chipand GeneChip Mouse Gene 1.0 ST Array Chip, respectively(Affymetrix, Santa Clara, CA), using the standard protocolsof the Genomic Core Facility, CABIMER. Image analysis,quality control, and data quantification were performedusing Affymetrix GeneChip Command Console version 4.0software. Additional statistical data analysis was per-formed using the Transcriptome Analysis Console (TAC)developed by Affymetrix. Modulated pathways using TACsoftware were obtained by applying significantly modu-lated genes and pathways with at least three modulatedgenes. The Ingenuity Pathway Analysis platform was usedto determine modulations on annotated canonical path-ways and annotated diseases and functions using thesignificantly modulated genes.

Patient Samples and Genetic AnalysisWomen in the second and third trimester of gestation wereinvited to participate in the genetic screening of PAX8 ifthey were diagnosed with GDM by OGTT per defined byinternational criteria or by HbA1c .6.5% (48 mmol/mol)(criterion 1) (15). Indexed patients were then selected onthe basis of hypothyroidism defined as free levothyroxine(FT4),0.90 ng/dL or thyroid stimulating hormone (TSH).2.5 mU/L during the first trimester of pregnancy orTSH .3 mU/L during the second to third trimesters ofpregnancy of nonautoimmune nature (absence of circulat-ing thyroglobulin [TG] and thyroperoxidase antibodies)(criterion 2). The rationale for this second criterion was toincrease the probability of identifying PAX8 mutations,a factor for which mutations are associated with thyroiddisorders. Exclusion criteria were 1) patients not takingiodine supplements and 2) patients with a BMI .30kg/m2.

In addition, accessible relatives of the index patient infamilial pedigree 1 (T356M) also were invited to partici-pate in the genetic screening. After obtaining informedconsent at Hospital La Paz, Madrid, using protocols ap-proved by the local ethics committee, blood was collectedand genomic DNA extracted from peripheral leukocytesusing the Chemagen DNA blood kit in the Chemagicautomated system (PerkinElmer). The entire coding se-quence of the human PAX8 gene (reference cDNA:NM_003466.3; Genome location chr2: 113992991) wasamplified by PCR under standard conditions. DNA se-quencing was performed in an ABI 370 sequencer andanalyzed with Sequencher software.

PAX8 Mutagenesis and Protein Stability StudiesSite-directed mutagenesis (GenScript, Piscataway Town-ship, NJ) was performed on the human PAX8 cDNA togenerate mutant variants PAX8-P25R and PAX8-T356M(16). These variants were subcloned into the pCDNA3.1vector, and the fidelity of the mutated constructs wasconfirmed by sequencing. To assess protein stability and

subcellular localization, MCF-7 cells that do not expressPAX8 (8) were transfected with the human PAX8Wt,T356M, and P25R using lipofectamine (Thermo FisherScientific) according to the manufacturer’s protocol. Twenty-four hours posttransfection, cells were either treatedor not with cycloheximide (80 mg/mL) for 24 and48 h. Protein extracts were then prepared and Westernblotting performed against PAX8. Alternatively, trans-fected cells were processed for immunofluorescence anal-ysis as previously described (12).

In Vitro Transactivation AssayThe human TG gene promoter, a PAX8 target, was subcl-oned into the firefly luciferase reported plasmid (17) andtransfected in HEK293T cells along with expression vec-tors for thyroid transcription factor 1 (TTF1 [also knownas NKX2.1]) in combination with PAX8Wt, T356M, orP25R using the FuGENE 6 reagent (Promega BiotechIbérica, Madrid, Spain). To correct for transfection effi-ciency, Renilla-encoding pRL-cytomegalovirus vector wascotransfected in all cases. Cellular extracts were collected48 h posttransfection, and Dual-Luciferase Reporter Assay(Promega) was performed according to the manufacturer’sinstructions. The ratio between the luciferase and Renillaactivities was expressed relative to the ratio obtained incells transfected with reporter and empty expressionvector (pCDNA3).

StatisticsData are shown as mean 6 SD. Paired and unpairedStudent t test, Wilcoxon rank sum test, Kruskal-Wallisone-way ANOVA, and one-way ANOVA were performedwhen appropiate using SigmaPlot 12.0 software (Sigma-Plot, Barcelona, Spain).

RESULTS

PAX8 Is Transiently Expressed in Pancreatic IsletsDuring PregnancyWe initially validated that PAX8 expression was transientlyinduced in mouse islets during pregnancy, reaching max-imal fold expression at pcd 14.5 and thereafter returningto basal levels by pcd 18.5 (Fig. 1A). As previously reported,Tph1 transcript levels also were increased, whereas thePdx1 transcript did not show significant alterations (4)(Fig. 1B and C). To extend these findings to human, iso-lated human islets were treated with recombinant humanprolactin to mimic conditions of the third trimester ofpregnancy (18). Prolactin produced a significant 2.5-foldincrease in PAX8 expression at 72 h of treatment comparedwith untreated islets (Fig. 1D). TPH1 expression also wasincreased, starting at 24 h of treatment, whereas nosignificant changes were observed in PDX1 expressionlevels (Fig. 1E and F). Prolactin treatment caused a rapidand transient increase in the proliferation rate of humanislet cells, peaking at 24 h of treatment (Fig. 1G). Of note,the PAX8 expression pattern inversely correlated withproliferation rates, suggesting that the transcription factor

diabetes.diabetesjournals.org Martin-Montalvo and Associates 111

is not involved in human islet cell replication. Taken to-gether, PAX8 expression can be induced in human isletsunder conditions mimicking pregnancy.

PAX8 Regulates Distinct Genetic Pathways in Mouseand Human Islets That Converge to Blunt ApoptosisTo establish the putative function of PAX8 in the endo-crine pancreas, we conducted global gene profiling ofisolated human andmouse islets transduced with lentiviralconstructs harboring either the human or the mouse PAX8cDNA within a bicistronic cassette with GFP. The rationaleof using both species-derived factors stemmed from ourprevious studies that revealed interspecies differencesbetween human and murine PAX4 (19). Flow cytometryanalysis demonstrated that 48 h after infection, ;75% ofislet cells expressed GFP (Fig. 2A and B). Principal com-ponent analysis (PCA) clearly separated islets overexpress-ing PAX8 from control-infected islets, illustrating therobust effect of PAX8 on overall gene expression (Fig.2C and D). Although several genes were similarly regulatedby PAX8 in both mouse and human islets (Fig. 2E), thefunctional enriched pathways were species specific (Fig. 2Fand G and Supplementary Fig. 1A and B). Of note, wedetermined that lentiviral-mediated murine Pax8 andhuman PAX8 overexpression resulted in a similar modu-lation of the most upregulated and downregulated tran-scripts in murine islets (Fig. 2H and Supplementary Tables4 and 5). Analysis of annotated “Disease and Functions”(Ingenuity Pathway Analysis module) confirmed that thetranscriptome profile of mouse islets overexpressing Pax8was associated predominantly with immune-related dis-eases, whereas the one from PAX8 overexpressing hu-man islets included immune-related diseases; endocrine/

metabolic disorders; and cell cycle, cell death, and cancer(Supplementary Fig. 1C and D and Supplementary Tables 6and 7). To analyze the functional effect of PAX8 in islets,we studied the effect of either murine or human PAX8overexpression in mouse islets. Functional cell metabolicactivity as well as GSIS were unaltered in murine isletstransduced with either the human or the mouse PAX8compared with mock-transduced islets (Fig. 2I and J). Ofnote, overexpression of mouse PAX8 in murine isletsreduced apoptosis by ;40% under basal conditions andblunted cytokine-induced apoptosis (Fig. 2K and L). Asimilar reduction also was confirmed by immunostainingthe apoptotic marker cleaved caspase-3 in transducedb-cells (Supplementary Fig. 2). Human PAX8 overexpres-sion in both mouse (Fig. 2K) and human islets (Fig. 2M)also significantly decreased by ;20% apoptosis. Collec-tively, these data suggest that both human and mousePAX8 promote islet cell survival in bothmurine and humanpancreatic islets.

Two PAX8 Mutations Are Found in Patients With GDMand Gestational Thyroid DysfunctionOur data demonstrating that PAX8 is transiently inducedin islets specifically during pregnancy combined with itsprosurvival properties provide strong arguments forPAX8 as a candidate gene for the study of GDM. Towardaddressing this possibility, we assessed the impact ofPax8 ablation in mouse on glucose homeostasis duringgestation. Because Pax82/2 pups die early postnatally (5)and are infertile despite thyroid hormone supplemen-tation (20,21), we worked with Pax8 heterozygous mice(Pax8+/2). Pax8+/2 females remained normoglycemic anddid not suffer glucose intolerance throughout pregnancy

Figure 1—PAX8 is transiently expressed in pancreatic islets during gestation. A–C: Determination of Pax8, Tph1, and Pdx1 mRNA levelsmeasured by real-time RT-PCR in murine islets at various stages of pregnancy. n = 4–5 animals per time point. D–F: Time coursedetermination of PAX8, TPH1, and PDX1 mRNA levels measured by real-time RT-PCR in human islets treated daily with prolactin(200 ng/mL). n = 8 per group at times 0, 24, 48, and 72 h and n = 7 at time 96 h in D. n = 4 per time point in E. n = 5 per group attimes 0, 24, 48, 72 h and n = 3 at time 96 h in F. G: Determination of proliferation by BrdU incorporation in human islets at various prolactintreatment time points. BrdU was added to the culture 24 h before processing the samples. n = 5–6 islet preparations per time point. Data aremean 6 SD of nonpregnant (NP) females or 0 h of treatment. *P , 0.05 vs. NP mice or time 0 in human islets. r.u., relative unit.

112 PAX8 and Pancreatic Islets in Pregnancy Diabetes Volume 68, January 2019

Figure 2—PAX8 targets different genetic pathways in human and mouse islets that converge in improved islet survival. Human and murinepancreatic islets were transduced with mock (GFP control), mouse Pax8, or human PAX8 lentiviral vectors. A: Flow cytometry analysisaccording to size (forward scattered light [FSC]) and granularity (side scattered light [SSC]). The delineated subpopulation was used todetermine transduction efficiency. B: Flow cytometry histograms showing the number of events plotted against GFP fluorescence. The lineindicates the threshold for positivity.C andD: DNAmicroarray profilingwas performed, and the PCAwas applied for data derived frommurineand human islets. Each point corresponds to the PCA analysis of one biological replicate. n = 3 independent replicates. E: Heatmap depictingthe relative expression levels of shared significantly modulated genes compared with control in murine and human pancreatic islets. F andG:Comparison of genetic pathways significantly altered by the overexpression of the murine Pax8 or the human PAX8 compared with mock-transduced islets using the TAC platform. H: Determination of gene expression on murine pancreatic islets infected with mock, Pax8, orPAX8. n = 3 biological replicates per experimental group. I: Determination of metabolic activity using the MTT assay in transduced murineislets. n = 4 per condition. J: Insulin secretion of transduced murine islets was assessed in 30-min static incubations in response to 2.5 or16.5 mmol/L glucose. Results are presented as a stimulation index (16 mmol/L/2.5 mmol/L glucose). n = 3–4 per condition. K–M: Apoptosiswas measured by ELISA in transducedmurine islets (n = 5–8 per group), murine islets challenged with cytokines (IFN-g 1,000 units/mL, IL-1b100 units/mL, and TNF-a 1,000 units/mL) (n = 3 per group), and human islets (n = 3 donors per group). One-way ANOVA shown in H and J.Kruskal-Wallis one-way ANOVA for ranks shown in I, K, and L. Wilcoxon rank sum test inM. *P , 0.05 compared with mock in H and mockchallenged with cytokines in L. GPCR, G-protein–coupled receptor; r.u., relative unit.

diabetes.diabetesjournals.org Martin-Montalvo and Associates 113

Figure 3—The PAX8-T356M and PAX8-P25R mutations are found in two patients who developed GDM, and both mutations exhibitcompromised transcriptional activity. A: Family pedigree 1 (T356M) with GDM as well as T2DM and hypothyroidism in consecutivegenerations. Circles indicate females, and squares indicate males. B: Sequencing chromatogram of T356M mutation in the index patient ofpedigree 1 and aWt sequence in a healthy relative. C: Schematic representation of PAX8 protein with annotated knownmutations, includingT356M and P25R. D–F: OGTT during pregnancy (weeks 24–28) in the index patient harboring T356M. Levels above the threshold of NDDGand/or CC diagnosis for GDM are highlighted in red. G: Family pedigree 2 (P25R) with GDM and hypothyroidism. H: OGTT during the firstpregnancy in index patient of pedigree 2 harboring P25R. I and J: MCF-7 cells were transfected with an empty pCDNA3.1, PAX8WtpCDNA3.1, PAX8-T356M pCDNA3.1 (T356M), or PAX8-P25R pCDNA3.1 (P25R) plasmid. Transfected cells were then incubated with80mg/mL cycloheximide (CHX) for the indicated time. Transfected PAX8 and endogenous b-actin protein levels were determined by standardimmunoblotting. n = 3 independent experiments performed in triplicate. I: Representative image of the immunoblots. J: Quantification of band

114 PAX8 and Pancreatic Islets in Pregnancy Diabetes Volume 68, January 2019

(Supplementary Fig. 3). However, the fact that thyroidhormone levels, for which biosynthesis is directly regulatedby PAX8, were similar in both Pax8+/2 and Pax8+/+ (Wt) micesuggests a potential genetic compensation for Pax8 hap-loinsufficiency (data not shown). Consequently, we optedto perform a genetic screening for mutations in the PAX8gene in patients with GDM and gestational thyroid dys-function (GTD) (for family pedigrees, clinical character-istics, and glucose tolerance tests during pregnancy, seeSupplementary Table 8 and Supplementary Figs. 4 and 5).The rationale of using patients with GTD, a disease asso-ciated with PAX8 mutations, was to optimize probabilitiesof identifying novel PAX8 gene mutations in a small co-hort. Consistent with this premise, exon sequencing ofPAX8 in members of seven pedigrees (pedigrees 1, 3, 4, 5,6, 7, and 8) identified a woman who belongs to pedigree1 and bears a novel heterozygous variant (c.1067C.T)(Fig. 3A and B). This missense mutation causes a threonine-to-methionine substitution at codon 356 (p.T356M) lo-cated at the carboxy-terminal region of the protein,distant from the majority of identified PAX8 mutationsthat are located within the DNA-binding paired domain(Fig. 3C). The index patient, a 30-year-old south occidentalEuropean Caucasian woman, was diagnosed with GTD in allfour pregnancies and was prescribed FT4 (25–50 mg/day)(Table 1). Although no data are available for the firstpregnancy, the patient exhibited impaired glucose toleranceafter OGTTs in the other three pregnancies and was di-agnosed with GDM according to National Diabetes DataGroup (NDDG) and Carpenter-Coustan (CC) guidelines(22,23) (Fig. 3D–F). She could control hyperglycemiathrough a carbohydrate-restricted diet and physical exer-cise. To date, the patient has not been diagnosed with DMunder nonpregnant conditions, and her HbA1c levels arenormal (Table 1). Her father, who also carries the muta-tion, was diagnosedwith T2DM (HbA1c 6.7% [50mmol/mol])at age 55 years (Fig. 3A). A 20-year-old sister of the indexpatient also harbors the mutation but has never beenpregnant. Of note, numerous family members of thepedigree who are deceased and therefore not accessiblefor genotyping also were diagnosed with T2DM and/ornonautoimmune hypothyroidism (Fig. 3A).

To rule out the possibility that the c.1067C.T variantis a common PAX8 polymorphism in the general popula-tion, we genotyped 100 control chromosomes of south-occidental European Caucasians. None of these individualspossessed this variant (minor allele frequency ,0.00034).Moreover, this mutation is not documented in the ExomeVariant Server (http://evs.gs.washington.edu/EVS), Database

of Single Nucleotide Polymorphisms (www.ncbi.nlm.nih.gov/snp), Exome Aggregation Consortium (http://exac.broadin-stitute.org), and Genome Aggregation Database (http://gno-mad.broadinstitute.org). In silico analysis using consensusclassifier prediction tools for disease-related mutationssupported a pathogenic role of p.T356M (24) (SupplementaryTable 9). Consequently, we tested whether the PAX8-T356Mmutation affected protein stability and/or subcellular lo-calization. For these studies, we included the PAX8-P25Rmutant variant (P25R, c.74C.G) associated with congen-ital hypothyroidism that lacks DNA binding activity butretains nuclear localization (16). Of note, a retrospectiveclinical analysis of clinical data and familial pedigree(pedigree 2) of the family harboring the P25R mutationrevealed that the only gestating female bearing this mu-tation also developed GDM (Fig. 3G and H and Supple-mentary Table 8). A recent second pregnancy resulted ina miscarriage at week 7. The stability of both mutantproteins was similar to that of PAX8Wt in transfectedMCF-7 cells treated with the protein synthesis inhibitorcycloheximide (Fig. 3I and J). Nuclear localization of themutant variants also was unaltered (Fig. 3K).

We then assessed the functional impact of PAX8-P25Rand PAX8-T356M mutations. To this end, we analyzedby luciferase reporter assay the transactivation potentialof PAX8Wt, PAX8-P25R, and PAX8-T356M on the TG genepromoter. The PAX8 cofactor TTF1 (NKX2.1) also wasincluded to determine whether these mutations have animpact on the synergistic transcriptional activation ob-served between the PAX8Wt and TTF1 (25). Accordingly,cotransfection of both PAX8Wt and TTF1 resulted ina marked increase in luciferase activity (Fig. 3L). In con-trast, addition of PAX8-T356M or PAX8-P25R along withTTF1 resulted in ;60% and ;35% of PAX8Wt and TTF1combined activity, respectively, establishing the compro-mised functionality of PAX8-T356M and PAX8-P25Rcompared with PAX8Wt (Fig. 3L).

DISCUSSION

We previously refuted that PAX8 is expressed either inmurine adult islets or in human pancreatic neuroendocrinetumors, which raises serious concerns about a potentialrole of this PAX family member in islet physiology in healthand disease (6). Notwithstanding, we report that PAX8 isinduced in human islets treated with the lactogenic hor-mone prolactin and that forced expression improves isletsurvival. Moreover, we identified a novel PAX8 missensemutation, p.T356M (pedigree 1), in a heterozygous female

intensities normalized to b-actin and plotted as a percentage of the PAX8Wt initial (time 0) band intensity. K: Representative immuno-fluorescence images depicting normal subcellular localization of the different PAX8 variants. n = 3 independent experiments performed intriplicate. Scale bars are 25 mm. Arrows indicate representative positive staining. L: Functional analysis of PAX8 variants. Hek293 cells weretransfected with empty pCDNA3.1 or PAX8-expressing pCDNA3.1 constructs along with human reporter plasmid TG promoter luciferasewith or without TTF1 pCDNA3.1. Luciferase activity is shown as fold change relative to the activity observed in the presence of reporter TTF1and PAX8Wt. n = 4 per experimental group. Data are mean6 SD. *P, 0.05 vs. PAX8Wt-transfected cells. NA, not available; r.u., relative unit.

diabetes.diabetesjournals.org Martin-Montalvo and Associates 115

Tab

le1—

Clin

ical

features

oftheindex

patient

ofped

igree1ha

rboring

PAX8-T35

6M

Pregn

ancy

1(28ye

arsold)

Pregn

ancy

2(30ye

arsold)

Pregn

ancy

3(31ye

arsold)

Pregn

ancy

4(33ye

arsold)

Normal

rang

eNP

Sho

rtOGTT

(O’Sullivan

protoc

ol)

++

++

ND

Long

OGTT

(NDDG

protoco

l)+

++

+ND

Bas

alinsu

lin(m

U/L)

ND

5ND

ND

3–25

3

HbA

1c

%ND

4.9–

5ND

ND

4–6.5

5.2

mmol/m

olND

30–31

ND

ND

20–48

33

GDM

diagn

osis

++

++

2

Res

pon

seto

trea

tmen

t(diet+ph

ysical

exercise

)+

++

+—

TSH

(mU/L)

NA

1T:2.92

2T:1.96

1T:3.29

2T:1.89

3T:1.49

1T:1.21

2T:3.2

3T:3.01

1T:,2.5

2T:,3

3T:,3

NP:0.27

–4.2

1.62

FT4(ng/mL)

NA

2T:1.2

2T:0.94

1T:0.95

0.93

–1.7

1.08

Serum

TG(ng/mL)

ND

ND

ND

1T:36

0.6–

6029

.6

GTD

diagn

osis

++

++

2

Levo

thyrox

inetrea

tmen

t(mg/day

)2T

:25

3T:25

2T:25

3T:25

2T:25

3T:50

1T:25

2T:25

3T:25

—

Abno

rmal

values

arehigh

lighted

inbold.1T

,firsttrim

ester;2T

,se

cond

trim

ester;3T

,third

trim

ester;NA,no

tav

ailable;ND,no

tdetermined

;NP,no

npregn

ant.

116 PAX8 and Pancreatic Islets in Pregnancy Diabetes Volume 68, January 2019

diagnosed with GDM and GTD, and a retrospective anal-ysis of clinical data indicates that a female harboring themutation PAX8-P25R (pedigree 2) developed GDM. Thus,our results highlight an unprecedented role for PAX8 inislet survival in response to a specific metabolic stress(e.g., pregnancy).

Although we found distinct transcriptome profiles be-tween murine and human islets overexpressing the re-spective species-specific PAX8, both converged to reduceislet apoptosis. In particular, during pregnancy, the pla-centa, among other tissues, is known to increase thesecretion of proinflammatory cytokines to induce periph-eral insulin resistance and favor the entrance of nutrientsto the fetus during gestation (4). Therefore, it is temptingto speculate that PAX8 could be involved in the protectionof pancreatic islets from the proinflammatory milieu thatoccurs during pregnancy as well as in adaptive mechanismsthat cope with the increased demand for insulin biosyn-thesis and secretion, contributing to prevent stress-induced cell death under these metabolic circumstances.In support of this premise, we found that lentiviral-mediated PAX8 overexpression reduces the apoptosis ratein both murine and human pancreatic islets while main-taining normalmetabolic activity and functionality. Of note,because Pax8 is expressed only transiently during preg-nancy, these beneficial effects are likely acute, which is inline with the concept that postpartum, b-cells undergoincreased apoptosis to restore functional mass to prepreg-nancy conditions (26). The antiapoptotic attributes ofPAX8 have been described in other cell types (27) andthe lack of Pax8 expression has been associated withincreased apoptotic death in the cardiomyocytes of Pax8homozygous knockout mice (28); therefore, our findingssuggest that specific interventions targeting PAX8 in pan-creatic endocrine cells could be explored to enhance isletcell survival.

The infertility of Pax82/2 mice treated with thyroidhormone has hampered the validation of Pax8 require-ment to maintain normoglycemia during pregnancy. How-ever, our assumption that PAX8 might act as a prosurvivalgene in islets specifically during gestation is supported bythe identification and functional characterization of anunprecedented PAX8mutation (p.T356M) found in a preg-nant female who developed GDM. This mutation also wasdetected in the index patient’s father who was diagnosedwith T2DM, suggesting a potential association of PAX8with T2DM as previously proposed in a genome-widelinkage and admixture mapping study (10). Of note,two sisters of the index patient also were genotyped for thePAX8 mutation. Although one young sister carries themutation T356M but has never been pregnant, the secondsister lacks the mutation and was diagnosed with GTD butnot GDM during pregnancy. These data suggest that onthis specific genetic background, which is prone to developthyroid complications as seen in the paternal and maternalsides of familial pedigree 1, the p.T356M variant maytrigger GDM. It will be imperative to monitor the youngest

sister for signs of GDM in her first pregnancy. In additionto the p.T356M mutation, the only pregnant female ofa familial pedigree harboring the mutation p.P25R de-veloped GDM and elevated HbA1c levels during pregnancy.Overall, although our results cannot be considered fullyconclusive with regard to the pathogeny of PAX8 muta-tions in the development of GDM and the study of largercohorts of patients harboring mutations on PAX8 is war-ranted, our data suggest that PAX8 could be considered asa candidate gene for the study of glucose metabolismdisorders during pregnancy.

To date, most PAX8 mutations associated with thyroiddysgenesis are located in the highly conserved DNA-bind-ing paired domain (29,30) (Fig. 3B). In contrast, p.T356Mis located within the carboxy-terminal of the protein thatwas shown to be essential for synergistic transactivation ofthe TG gene promoter along with the homeodomain-containing transcription factor TTF1 (25). Accordingly,although the capacity of DNA binding might not becompromised in the PAX8-T356M variant, its synergistictranscriptional activity along with TTF1 was reducedcompared with PAX8Wt, suggesting that the mutationalters protein-protein interactions. As such, the PAX8-T356M variant may exert a dominant-negative effect,which may render a more severe phenotype comparedwith other mutations that could either impede DNAbinding or generate a truncated nonfunctional variantof the protein, as observed in Pax8 heterozygous mice.

In conclusion, our data indicate that PAX8 expressionin pancreatic islets during pregnancy might favor isletsurvival under this challenging metabolic condition. Fur-thermore, PAX8 could be considered as a candidate genefor the study of GDM.

Acknowledgments. The authors acknowledge the full collaboration ofpatients and their family members in the study. The authors thank Isabel Moreno-Navarro and Mercedes Tanarro (Thyroid Molecular Laboratory, Institute for Medicaland Molecular Genetics, La Paz University Hospital, Autonomous University ofMadrid, Madrid, Spain) for excellent technical work. The authors also acknowledgethe computer resources provided by the Andalusia Bioinformatics Platform.Funding. This work was funded by grants from the Ministerio de Economía yCompetitividad, Instituto de Salud Carlos III, co-funded by fondos FEDER(CP14/00105 and PI15/00134 to A.M.-M., PI16/00830 to J.C.M., andPI13/00593 and BFU2017-83588-P to B.R.G.). Human islets were procuredthrough the European Consortium for Islet Transplantation funded by JDRF (3-RSC-2016-162-I-X).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. A.M.-M., L.L.-N., C.J.-M., A.H., P.I.L., N.C.-V.,A.T., C.G.-G., J.S.W.R.H., F.L., D.B., M.G.T., L.H., J.A., J.C.M., and B.R.G.discussed the results and commented on the manuscript. A.M.-M., L.L.-N.,C.J.-M., P.I.L., N.C.-V., and M.G.T. performed the experiments using pancreaticislets, MCF-7 cells, and site-directed mutagenesis. A.M.-M., J.C.M., and B.R.G.designed and supervised the study, secured funding, analyzed the data, and wrotethe manuscript. A.H., A.T., C.G.-G., J.S.W.R.H., L.H., and J.C.M. designed andperformed human studies and transactivation assays. F.L. and D.B. providedhuman pancreatic islets. J.A. performed the human studies related to the P25Rvariant. B.R.G. is the guarantor of this work and, as such, had full access to all the

diabetes.diabetesjournals.org Martin-Montalvo and Associates 117

data in the study and takes responsibility for the integrity of the data and theaccuracy of the data analysis.Data Availability. The Microarray raw data on pancreatic islets over-expressing the murine or human PAX8 have been deposited in the GeneExpression Omnibus repository with the accession number GSE94846.

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118 PAX8 and Pancreatic Islets in Pregnancy Diabetes Volume 68, January 2019