MARTIN-MONTALVO, Alejandro, et al .

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Reference

Transient PAX8 Expression in Islets During Pregnancy Correlates With β-Cell Survival, Revealing a Novel Candidate Gene in

Gestational Diabetes Mellitus

MARTIN-MONTALVO, Alejandro, et al .

Abstract

Transient Pax8 expression was reported in mouse islets during gestation, whereas a genome-wide linkage and admixture mapping study highlighted PAX8 as a candidate gene for diabetes mellitus (DM). We sought the significance of PAX8 expression in mouse and human islet biology. PAX8 was induced in gestating mouse islets and in human islets treated with recombinant prolactin. Global gene expression profiling of human and mouse islets overexpressing the corresponding species-specific PAX8 revealed the modulation of distinct genetic pathways that converge on cell survival. Accordingly, apoptosis was reduced in PAX8-overexpressing islets. These findings support that PAX8 could be a candidate gene for the study of gestational DM (GDM). PAX8 was genotyped in patients with GDM and gestational thyroid dysfunction (GTD), a pathology commonly found in patients with mutations on PAX8 A novel missense PAX8 mutation (p.T356M, c.1067C>T) was identified in a female diagnosed with GDM and GTD as well as in her father with type 2 DM but was absent in control patients. The p.T356M variant did not alter protein stability or cellular [...]

MARTIN-MONTALVO, Alejandro, et al . Transient PAX8 Expression in Islets During Pregnancy Correlates With β-Cell Survival, Revealing a Novel Candidate Gene in Gestational Diabetes Mellitus. Diabetes , 2019, vol. 68, no. 1, p. 109-118

DOI : 10.2337/db18-0285 PMID : 30352879

Available at:

http://archive-ouverte.unige.ch/unige:138454

Disclaimer: layout of this document may differ from the published version.

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Transient PAX8 Expression in Islets During Pregnancy Correlates With b -Cell Survival, Revealing a Novel

Candidate Gene in Gestational Diabetes Mellitus

Alejandro 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,²Fanny Lebreton,³Domenico Bosco,³Miguel García Toscano,⁴Lucrecia 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

TransientPax8expression was reported in mouse islets during gestation, whereas a genome-wide linkage and admixture mapping study highlightedPAX8as a candidate gene for diabetes mellitus (DM). We sought the signiﬁcance of PAX8 expression in mouse and human islet biology.PAX8was induced in gestating mouse islets and in human islets treated with recombinant prolactin.

Global gene expression proﬁling of human and mouse islets overexpressing the corresponding species- speciﬁc PAX8 revealed the modulation of distinct genetic pathways that converge on cell survival. Accordingly, apoptosis was reduced in PAX8-overexpressing islets.

Theseﬁndings support thatPAX8could be a candidate gene for the study of gestational DM (GDM).PAX8was genotyped in patients with GDM and gestational thyroid dysfunction (GTD), a pathology commonly found in patients with mutations on PAX8. A novel missense PAX8 mutation (p.T356M, c.1067C>T) was identiﬁed in a female diagnosed with GDM and GTD as well as in her father with type 2 DM but was absent in control patients.

The p.T356M variant did not alter protein stability or cellular localization, whereas its transactivation activity was hindered. In parallel, a retrospective clinical analysis uncovered that a pregnant female harboring a second PAX8mutation (p.P25R, c.74C>G) previously reported to cause congenital hypothyroidism also developed GDM.

These data indicate that increased expression of PAX8 affects islet viability and thatPAX8could be considered as a candidate gene for the study of GDM.

During gestation, maternal metabolic adaptations are essential to ensure the health of the mother and the viability of the fetus. One of the organs that must undergo a pro- found adaptation during pregnancy is the endocrine pancreas. The isletb-cell mass expands as an adaptive response to the progressive insulin resistance that devel- ops in the pregnant female to favor nutrient accessibility to the fetus (1). This mechanism ofb-cell mass expansion includesb-cell hypertrophy, increasedb-cell proliferation, and increased prosurvival/antiapoptotic signaling (2). Fail- ure to adapt may result in the development of gestational diabetes mellitus (GDM), with concomitant health issues not only for the mother but also for the fetus. The under- lying molecular mechanisms triggering GDM remain largely uncharted (3). Toward identifying these molecular pathways, two independent studies have analyzed the transcriptome landscape of islets isolated from gestating mice (2,4). Of note, one of the most upregulated genes was 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, Spain

2Thyroid Molecular Laboratory, Institute for Medical and Molecular Genetics, La Paz University Hospital, Autonomous University of Madrid, Madrid, Spain

3Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals, Geneva, Switzerland

4Amarna Therapeutics, Seville, Spain

5Department of Endocrinology and Nutrition, Hospital Divino Espírito Santo, Ponta Delgada, Portugal

Corresponding author: Benoit R. Gauthier, benoit.gauthier@cabimer.es;

Alejandro Martin-Montalvo, alejandro.martinmontalvo@cabimer.es; or José Carlos 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 as long as the work is properly cited, the use is educational and not for proﬁt, and the work is not altered. More information is available at http://www.diabetesjournals .org/content/license.

Diabetes Volume 68, January 2019 109

ISLETSTUDIES

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development and function of the thyroid and urogenital system, maintaining its expression in adult kidney and thyroid gland (5). In agreement with this described pattern of expression, mutations/polymorphisms in PAX8 have been associated with hypothyroidism and urorenal abnormalities, whereas its overexpression correlates with tumor formation in these and other tissues (6,7).

We have previously reported that Pax8 is expressed neither during mouse pancreatic development nor in adult islets under normal physiological conditions (8,9). This fact raises the prospect that PAX8 expression likely will be induced transiently under specific physiological conditions, such as pregnancy, which may confer benefits to islets. Consistent with this beneficial effect of PAX8 under situations of increased insulin demand, a single genome- wide linkage and admixture mapping study has high- lightedPAX8 as a putative type 2 diabetes mellitus (T2DM) candidate gene in African American descendants (10). In the current study, we sought to validate PAX8 expression and determine its physiological function during pregnancy. We observed that PAX8 is transiently expressed in mouse under gestating conditions as well as in human islets 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 ofPAX8 (p.T356M, c.1067C.T) in a family pedigree in which a female developed GDM while her father developed T2DM. Functional analysis demonstrated that the p.T356M mutation signiﬁcantly decreased PAX8 transactivation potential. In addition, a retrospective clinical analysis uncovered that GDM developed in a pregnant female of a familial pedigree that harbors a previously described PAX8-P25R mutation associated with congenital hypothyroidism.

RESEARCH DESIGN AND METHODS Mice

C57BL/6J mice were purchased from JANVIER LABS (Saint-Berthevin, France). Five-month-old C57BL/6J pregnant females were euthanized at post coitum day (pcd) 10.5, 14.5, 16.5, and 18.5 for islet isolation.Pax8knockout mice were procured from the European Mouse Mutant Archive network. Mouse experiments were approved by the Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER) animal committee and performed in accordance with Spanish law on animal use (RD 53/2013).

Oral Glucose Tolerance Test

Pregnant wild-type (Wt) and heterozygous Pax8 mice were subjected to an oral glucose tolerance test (OGTT) at pcd 14.5 as previously reported (11).

Pancreatic Islet Procurement and Treatment

Mouse 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 of human islet preparations are presented in Supplementary Table 1. Islets were cultured as previously described (14).

Where indicated, murine islets were treated for 16 h with cytokines at 48 h after lentiviral infection (interferon-g [IFN-g 1,000 units/mL, interleukin-1b [IL-1b 100 units/mL, and tumor necrosis factor-a [TNF-a 1,000 units/mL). Where indicated, human islets were treated with recombinant human prolactin (200 ng/mL daily) for up 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 remove hormonal contaminants.

Quantitative RT-PCR

Total RNA was extracted using the RNeasy Micro Kit (QIAGEN). cDNA using 0.5–1 mg RNA was synthe- sized using SuperScript II Reverse Transcriptase (Thermo Fisher Scientiﬁc, Madrid, Spain). The RT-PCR was performed on individual cDNAs using SYBR Green (Roche Diagnostics, Mannheim, Germany). Primer sequences are presented in Supplementary Table 2. The mRNA expression was calculated by the 2^2DDCTmethod and normalized to the expression ofcyclophilin,RPS29, and/orb-actin(12).

Generation of PAX8 Lentiviruses and Transduction The 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 control of the spleen focus-forming virus promoter while the constitutive ubiquitin promoter regulates expression of the reporter green fluorescent protein (GFP). Lentivirus amplification, purification, and transduction were performed as previously described (13). Islet transduction efficiency was estimated byflow cytometry (FACSCalibur;

BD Biosciences, Madrid, Spain) using ;20 disaggregated islets.

Islet Viability, Proliferation, and Insulin Secretion Assessment

Apoptosis and proliferation were assessed using cell death detection and BrdU cell proliferation ELISA, respectively (catalog #11647229001; Sigma-Aldrich, Madrid, Spain).

BrdU (10mmol/L) was added during the last 24 h of the assay at each time point. Glucose-stimulated insulin secretion (GSIS) was performed as previously described (14).

Immunohistochemistry

Islet embedding and immunohistochemistry analysis were performed as previously described (12). A list of primary and secondary antibodies is provided in Supplementary Table 3. Images were acquired using a Leica microscope (AF6000; Leica, Milton Keynes, U.K.).

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Transcriptome Proﬁling

cRNA preparations from human and murine islets were hybridized to GeneChip Human Gene 2.0 ST Array Chip and GeneChip Mouse Gene 1.0 ST Array Chip, respectively (Affymetrix, Santa Clara, CA), using the standard protocols of the Genomic Core Facility, CABIMER. Image analysis, quality control, and data quantification were performed using Affymetrix GeneChip Command Console version 4.0 software. Additional statistical data analysis was performed using the Transcriptome Analysis Console (TAC) developed by Affymetrix. Modulated pathways using TAC software were obtained by applying significantly modulated genes and pathways with at least three modulated genes. The Ingenuity Pathway Analysis platform was used to determine modulations on annotated canonical pathways and annotated diseases and functions using the significantly modulated genes.

Patient Samples and Genetic Analysis

Women in the second and third trimester of gestation were invited to participate in the genetic screening ofPAX8if they were diagnosed with GDM by OGTT per defined by international criteria or by HbA1c.6.5% (48 mmol/mol) (criterion 1) (15). Indexed patients were then selected on the 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 or TSH.3 mU/L during the second to third trimesters of pregnancy of nonautoimmune nature (absence of circulat- ing thyroglobulin [TG] and thyroperoxidase antibodies) (criterion 2). The rationale for this second criterion was to increase the probability of identifying PAX8 mutations, a factor for which mutations are associated with thyroid disorders. Exclusion criteria were1) patients not taking iodine supplements and 2) patients with a BMI .30 kg/m².

In addition, accessible relatives of the index patient in familial pedigree 1 (T356M) also were invited to participate in the genetic screening. After obtaining informed consent at Hospital La Paz, Madrid, using protocols approved by the local ethics committee, blood was collected and genomic DNA extracted from peripheral leukocytes using the Chemagen DNA blood kit in the Chemagic automated system (PerkinElmer). The entire coding sequence of the human PAX8 gene (reference cDNA:

NM_003466.3; Genome location chr2: 113992991) was ampliﬁed by PCR under standard conditions. DNA sequencing was performed in an ABI 370 sequencer and analyzed with Sequencher software.

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

subcellular localization, MCF-7 cells that do not express PAX8 (8) were transfected with the human PAX8Wt, T356M, and P25R using lipofectamine (Thermo Fisher Scientiﬁc) according to the manufacturer’s protocol. Twenty- four hours posttransfection, cells were either treated or not with cycloheximide (80 mg/mL) for 24 and 48 h. Protein extracts were then prepared and Western blotting performed against PAX8. Alternatively, transfected cells were processed for immunoﬂuorescence analysis as previously described (12).

In Vitro Transactivation Assay

The human TG gene promoter, a PAX8 target, was subcloned into thefirefly luciferase reported plasmid (17) and transfected in HEK293T cells along with expression vectors for thyroid transcription factor 1 (TTF1 [also known as NKX2.1]) in combination with PAX8Wt, T356M, or P25R using the FuGENE 6 reagent (Promega Biotech Ibérica, Madrid, Spain). To correct for transfection efficiency, Renilla-encoding pRL-cytomegalovirus vector was cotransfected in all cases. Cellular extracts were collected 48 h posttransfection, and Dual-Luciferase Reporter Assay (Promega) was performed according to the manufacturer’s instructions. The ratio between the luciferase andRenilla activities was expressed relative to the ratio obtained in cells transfected with reporter and empty expression vector (pCDNA3).

Statistics

Data are shown as mean 6 SD. Paired and unpaired Student t test, Wilcoxon rank sum test, Kruskal-Wallis one-way ANOVA, and one-way ANOVA were performed when appropiate using SigmaPlot 12.0 software (Sigma- Plot, Barcelona, Spain).

RESULTS

PAX8Is Transiently Expressed in Pancreatic Islets During Pregnancy

We initially validated thatPAX8expression was transiently induced in mouse islets during pregnancy, reaching max- imal fold expression at pcd 14.5 and thereafter returning to basal levels by pcd 18.5 (Fig. 1A). As previously reported, Tph1 transcript levels also were increased, whereas the Pdx1 transcript did not show significant alterations (4) (Fig. 1BandC). To extend thesefindings to human, isolated human islets were treated with recombinant human prolactin to mimic conditions of the third trimester of pregnancy (18). Prolactin produced a significant 2.5-fold increase inPAX8expression at 72 h of treatment compared with untreated islets (Fig. 1D).TPH1expression also was increased, starting at 24 h of treatment, whereas no significant changes were observed in PDX1 expression levels (Fig. 1EandF). Prolactin treatment caused a rapid and transient increase in the proliferation rate of human islet cells, peaking at 24 h of treatment (Fig. 1G). Of note, the PAX8 expression pattern inversely correlated with proliferation rates, suggesting that the transcription factor

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

PAX8 Regulates Distinct Genetic Pathways in Mouse and Human Islets That Converge to Blunt Apoptosis To establish the putative function of PAX8 in the endocrine pancreas, we conducted global gene proﬁling of isolated human and mouse islets transduced with lentiviral constructs harboring either the human or the mouse PAX8 cDNA within a bicistronic cassette with GFP. The rationale of using both species-derived factors stemmed from our previous studies that revealed interspecies differences between human and murine PAX4 (19). Flow cytometry analysis demonstrated that 48 h after infection,;75% of islet cells expressed GFP (Fig. 2AandB). Principal com- ponent analysis (PCA) clearly separated islets overexpressing PAX8 from control-infected islets, illustrating the robust effect of PAX8 on overall gene expression (Fig.

2CandD). Although several genes were similarly regulated by PAX8 in both mouse and human islets (Fig. 2E), the functional enriched pathways were species specific (Fig. 2F and G and Supplementary Fig. 1A and B). Of note, we determined that lentiviral-mediated murine Pax8 and human PAX8 overexpression resulted in a similar modulation of the most upregulated and downregulated tran- scripts in murine islets (Fig. 2Hand Supplementary Tables 4 and 5). Analysis of annotated “Disease and Functions” (Ingenuity Pathway Analysis module) confirmed that the transcriptome profile of mouse islets overexpressing Pax8 was associated predominantly with immune-related diseases, whereas the one from PAX8 overexpressing human islets included immune-related diseases; endocrine/

metabolic disorders; and cell cycle, cell death, and cancer (Supplementary Fig. 1CandDand Supplementary Tables 6 and 7). To analyze the functional effect of PAX8 in islets, we studied the effect of either murine or human PAX8 overexpression in mouse islets. Functional cell metabolic activity as well as GSIS were unaltered in murine islets transduced with either the human or the mouse PAX8 compared with mock-transduced islets (Fig. 2IandJ). Of note, overexpression of mouse PAX8 in murine islets reduced apoptosis by ;40% under basal conditions and blunted cytokine-induced apoptosis (Fig. 2K and L). A similar reduction also was conﬁrmed by immunostaining the apoptotic marker cleaved caspase-3 in transduced b-cells (Supplementary Fig. 2). Human PAX8 overexpression 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 mouse PAX8 promote islet cell survival in both murine and human pancreatic islets.

TwoPAX8Mutations Are Found in Patients With GDM and Gestational Thyroid Dysfunction

Our data demonstrating thatPAX8is transiently induced in islets speciﬁcally during pregnancy combined with its prosurvival properties provide strong arguments for PAX8as a candidate gene for the study of GDM. Toward addressing this possibility, we assessed the impact of Pax8 ablation in mouse on glucose homeostasis during gestation. BecausePax8²^/²pups die early postnatally (5) and are infertile despite thyroid hormone supplemen- tation (20,21), we worked with Pax8 heterozygous mice (Pax8^+/²).Pax8^+/²females remained normoglycemic and did not suffer glucose intolerance throughout pregnancy

Figure 1—PAX8is transiently expressed in pancreatic islets during gestation.A–C: Determination ofPax8,Tph1, andPdx1mRNA levels measured by real-time RT-PCR in murine islets at various stages of pregnancy.n= 4–5 animals per time point.D–F: Time course determination 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 andn= 7 at time 96 h inD.n= 4 per time point inE.n= 5 per group at times 0, 24, 48, 72 h andn= 3 at time 96 h inF.G: Determination of proliferation by BrdU incorporation in human islets at various prolactin treatment time points. BrdU was added to the culture 24 h before processing the samples.n= 5–6 islet preparations per time point. Data are mean6SD 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.

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Figure 2—PAX8 targets different genetic pathways in human and mouse islets that converge in improved islet survival. Human and murine pancreatic islets were transduced with mock (GFP control), mouse Pax8, or human PAX8 lentiviral vectors.A: Flow cytometry analysis according to size (forward scattered light [FSC]) and granularity (side scattered light [SSC]). The delineated subpopulation was used to determine transduction efficiency.B: Flow cytometry histograms showing the number of events plotted against GFPfluorescence. The line indicates the threshold for positivity.CandD: DNA microarray profiling was performed, and the PCA was applied for data derived from murine and human islets. Each point corresponds to the PCA analysis of one biological replicate.n= 3 independent replicates.E: Heat map depicting the relative expression levels of shared significantly modulated genes compared with control in murine and human pancreatic islets.FandG: 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, or PAX8.n= 3 biological replicates per experimental group.I: Determination of metabolic activity using the MTT assay in transduced murine islets.n= 4 per condition.J: Insulin secretion of transduced murine islets was assessed in 30-min static incubations in response to 2.5 or 16.5 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: Apoptosis was measured by ELISA in transduced murine islets (n= 5–8 per group), murine islets challenged with cytokines (IFN-g1,000 units/mL, IL-1b 100 units/mL, and TNF-a1,000 units/mL) (n= 3 per group), and human islets (n= 3 donors per group). One-way ANOVA shown inHandJ. Kruskal-Wallis one-way ANOVA for ranks shown inI,K, andL. Wilcoxon rank sum test inM. *P,0.05 compared with mock inHand mock challenged with cytokines inL. GPCR, G-protein–coupled receptor; r.u., relative unit.

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Figure 3—The PAX8-T356M and PAX8-P25R mutations are found in two patients who developed GDM, and both mutations exhibit compromised transcriptional activity. A: Family pedigree 1 (T356M) with GDM as well as T2DM and hypothyroidism in consecutive generations. Circles indicate females, and squares indicate males.B: Sequencing chromatogram of T356M mutation in the index patient of pedigree 1 and a Wt sequence in a healthy relative.C: Schematic representation of PAX8 protein with annotated known mutations, including T356M and P25R.D–F: OGTT during pregnancy (weeks 24–28) in the index patient harboring T356M. Levels above the threshold of NDDG and/or CC diagnosis for GDM are highlighted in red.G: Family pedigree 2 (P25R) with GDM and hypothyroidism.H: OGTT during theﬁrst pregnancy in index patient of pedigree 2 harboring P25R.IandJ: MCF-7 cells were transfected with an empty pCDNA3.1, PAX8Wt pCDNA3.1, PAX8-T356M pCDNA3.1 (T356M), or PAX8-P25R pCDNA3.1 (P25R) plasmid. Transfected cells were then incubated with 80mg/mL cycloheximide (CHX) for the indicated time. Transfected PAX8 and endogenousb-actin protein levels were determined by standard immunoblotting.n= 3 independent experiments performed in triplicate.I: Representative image of the immunoblots.J: Quantiﬁcation of band

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(Supplementary Fig. 3). However, the fact that thyroid hormone levels, for which biosynthesis is directly regulated by PAX8, were similar in bothPax8^+/²andPax8^+/+(Wt) mice suggests a potential genetic compensation forPax8 hap- loinsufﬁciency (data not shown). Consequently, we opted to perform a genetic screening for mutations in thePAX8 gene in patients with GDM and gestational thyroid dysfunction (GTD) (for family pedigrees, clinical characteristics, and glucose tolerance tests during pregnancy, see Supplementary Table 8 and Supplementary Figs. 4 and 5).

The rationale of using patients with GTD, a disease associated withPAX8mutations, was to optimize probabilities of identifying novelPAX8 gene mutations in a small cohort. Consistent with this premise, exon sequencing of PAX8in members of seven pedigrees (pedigrees 1, 3, 4, 5, 6, 7, and 8) identified a woman who belongs to pedigree 1 and bears a novel heterozygous variant (c.1067C.T) (Fig. 3AandB). This missense mutation causes a threonine- to-methionine substitution at codon 356 (p.T356M) located at the carboxy-terminal region of the protein, distant from the majority of identified PAX8 mutations that are located within the DNA-binding paired domain (Fig. 3C). The index patient, a 30-year-old south occidental European Caucasian woman, was diagnosed with GTD in all four pregnancies and was prescribed FT4 (25–50mg/day) (Table 1). Although no data are available for the first pregnancy, the patient exhibited impaired glucose tolerance after OGTTs in the other three pregnancies and was diagnosed with GDM according to National Diabetes Data Group (NDDG) and Carpenter-Coustan (CC) guidelines (22,23) (Fig. 3D–F). She could control hyperglycemia through a carbohydrate-restricted diet and physical exer- cise. To date, the patient has not been diagnosed with DM under nonpregnant conditions, and her HbA1c levels are normal (Table 1). Her father, who also carries the mutation, was diagnosed with T2DM (HbA1c6.7% [50 mmol/mol]) at age 55 years (Fig. 3A). A 20-year-old sister of the index patient also harbors the mutation but has never been pregnant. Of note, numerous family members of the pedigree who are deceased and therefore not accessible for genotyping also were diagnosed with T2DM and/or nonautoimmune hypothyroidism (Fig. 3A).

To rule out the possibility that the c.1067C.T variant is a commonPAX8 polymorphism in the general population, we genotyped 100 control chromosomes of south- occidental European Caucasians. None of these individuals possessed this variant (minor allele frequency,0.00034).

Moreover, this mutation is not documented in the Exome Variant Server (http://evs.gs.washington.edu/EVS), Database

of Single Nucleotide Polymorphisms (www.ncbi.nlm.nih.gov/

snp), Exome Aggregation Consortium (http://exac.broadinstitute.org), and Genome Aggregation Database (http://gno- mad.broadinstitute.org). In silico analysis using consensus classiﬁer prediction tools for disease-related mutations supported a pathogenic role of p.T356M (24) (Supplementary Table 9). Consequently, we tested whether the PAX8-T356M mutation affected protein stability and/or subcellular localization. For these studies, we included the PAX8-P25R mutant variant (P25R, c.74C.G) associated with congenital hypothyroidism that lacks DNA binding activity but retains nuclear localization (16). Of note, a retrospective clinical analysis of clinical data and familial pedigree (pedigree 2) of the family harboring the P25R mutation revealed that the only gestating female bearing this mutation also developed GDM (Fig. 3G and H and Supple- mentary Table 8). A recent second pregnancy resulted in a miscarriage at week 7. The stability of both mutant proteins was similar to that of PAX8Wt in transfected MCF-7 cells treated with the protein synthesis inhibitor cycloheximide (Fig. 3IandJ). Nuclear localization of the mutant variants also was unaltered (Fig. 3K).

We then assessed the functional impact of PAX8-P25R and PAX8-T356M mutations. To this end, we analyzed by luciferase reporter assay the transactivation potential of PAX8Wt, PAX8-P25R, and PAX8-T356M on theTGgene promoter. The PAX8 cofactor TTF1 (NKX2.1) also was included to determine whether these mutations have an impact on the synergistic transcriptional activation observed between the PAX8Wt and TTF1 (25). Accordingly, cotransfection of both PAX8Wt and TTF1 resulted in a marked increase in luciferase activity (Fig. 3L). In contrast, addition of PAX8-T356M or PAX8-P25R along with TTF1 resulted in;60% and;35% of PAX8Wt and TTF1 combined activity, respectively, establishing the compromised functionality of PAX8-T356M and PAX8-P25R compared with PAX8Wt (Fig. 3L).

DISCUSSION

We previously refuted that PAX8 is expressed either in murine adult islets or in human pancreatic neuroendocrine tumors, which raises serious concerns about a potential role of this PAX family member in islet physiology in health and disease (6). Notwithstanding, we report thatPAX8is induced in human islets treated with the lactogenic hormone prolactin and that forced expression improves islet survival. Moreover, we identiﬁed a novelPAX8 missense mutation, p.T356M (pedigree 1), in a heterozygous female

intensities normalized tob-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 in triplicate. Scale bars are 25mm. Arrows indicate representative positive staining.L: Functional analysis of PAX8 variants. Hek293 cells were transfected with empty pCDNA3.1 or PAX8-expressing pCDNA3.1 constructs along with human reporter plasmid TG promoter luciferase with or without TTF1 pCDNA3.1. Luciferase activity is shown as fold change relative to the activity observed in the presence of reporter TTF1 and PAX8Wt.n= 4 per experimental group. Data are mean6SD. *P,0.05 vs. PAX8Wt-transfected cells. NA, not available; r.u., relative unit.

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Table1—Clinicalfeaturesoftheindexpatientofpedigree1harboringPAX8-T356M Pregnancy1 (28yearsold)Pregnancy2 (30yearsold)Pregnancy3 (31yearsold)Pregnancy4 (33yearsold)NormalrangeNP ShortOGTT(O’Sullivanprotocol)++++ND LongOGTT(NDDGprotocol)++++ND Basalinsulin(mU/L)ND5NDND3–253 HbA1c %ND4.9–5NDND4–6.55.2 mmol/molND30–31NDND20–4833 GDMdiagnosis++++2 Responsetotreatment(diet+physicalexercise)++++— TSH(mU/L)NA1T:2.92 2T:1.961T: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)NA2T:1.22T:0.941T:0.950.93–1.71.08 SerumTG(ng/mL)NDNDND1T:360.6–6029.6 GTDdiagnosis++++2 Levothyroxinetreatment(mg/day)2T:25 3T:252T:25 3T:252T:25 3T:501T:25 2T:25 3T:25

— Abnormalvaluesarehighlightedinbold.1T,ﬁrsttrimester;2T,secondtrimester;3T,thirdtrimester;NA,notavailable;ND,notdetermined;NP,nonpregnant.

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

Although we found distinct transcriptome profiles between murine and human islets overexpressing the re- spective species-specific PAX8, both converged to reduce islet apoptosis. In particular, during pregnancy, the pla- centa, among other tissues, is known to increase the secretion of proinflammatory cytokines to induce peripheral insulin resistance and favor the entrance of nutrients to the fetus during gestation (4). Therefore, it is tempting to speculate that PAX8 could be involved in the protection of pancreatic islets from the proinflammatory milieu that occurs during pregnancy as well as in adaptive mechanisms that cope with the increased demand for insulin biosynthesis 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 rate in both murine and human pancreatic islets while maintaining normal metabolic activity and functionality. Of note, because Pax8 is expressed only transiently during pregnancy, these beneficial effects are likely acute, which is in line with the concept that postpartum, b-cells undergo increased apoptosis to restore functional mass to prepreg- nancy conditions (26). The antiapoptotic attributes of PAX8 have been described in other cell types (27) and the lack of Pax8 expression has been associated with increased apoptotic death in the cardiomyocytes ofPax8 homozygous knockout mice (28); therefore, our findings suggest that specific interventions targeting PAX8 in pancreatic endocrine cells could be explored to enhance islet cell survival.

The infertility of Pax8^2/2 mice treated with thyroid hormone has hampered the validation of Pax8 require- ment to maintain normoglycemia during pregnancy. How- ever, our assumption thatPAX8might act as a prosurvival gene in islets specifically during gestation is supported by the identification and functional characterization of an unprecedentedPAX8mutation (p.T356M) found in a pregnant female who developed GDM. This mutation also was detected in the index patient’s father who was diagnosed with T2DM, suggesting a potential association of PAX8 with T2DM as previously proposed in a genome-wide linkage and admixture mapping study (10). Of note, two sisters of the index patient also were genotyped for the PAX8 mutation. Although one young sister carries the mutation T356M but has never been pregnant, the second sister lacks the mutation and was diagnosed with GTD but not GDM during pregnancy. These data suggest that on this specific genetic background, which is prone to develop thyroid complications as seen in the paternal and maternal sides of familial pedigree 1, the p.T356M variant may trigger GDM. It will be imperative to monitor the youngest

sister for signs of GDM in herﬁrst pregnancy. In addition to the p.T356M mutation, the only pregnant female of a familial pedigree harboring the mutation p.P25R developed GDM and elevated HbA1clevels during pregnancy.

Overall, although our results cannot be considered fully conclusive with regard to the pathogeny of PAX8muta- tions in the development of GDM and the study of larger cohorts of patients harboring mutations onPAX8is war- ranted, our data suggest thatPAX8could be considered as a candidate gene for the study of glucose metabolism disorders during pregnancy.

To date, mostPAX8mutations associated with thyroid dysgenesis are located in the highly conserved DNA-binding paired domain (29,30) (Fig. 3B). In contrast, p.T356M is located within the carboxy-terminal of the protein that was shown to be essential for synergistic transactivation of the TG gene promoter along with the homeodomain- containing transcription factor TTF1 (25). Accordingly, although the capacity of DNA binding might not be compromised in the PAX8-T356M variant, its synergistic transcriptional activity along with TTF1 was reduced compared with PAX8Wt, suggesting that the mutation alters protein-protein interactions. As such, the PAX8- T356M variant may exert a dominant-negative effect, which may render a more severe phenotype compared with other mutations that could either impede DNA binding or generate a truncated nonfunctional variant of the protein, as observed inPax8heterozygous mice.

In conclusion, our data indicate thatPAX8expression in pancreatic islets during pregnancy might favor islet survival under this challenging metabolic condition. Fur- thermore,PAX8 could be considered as a candidate gene for the study of GDM.

Acknowledgments.The authors acknowledge the full collaboration of patients and their family members in the study. The authors thank Isabel Moreno- Navarro and Mercedes Tanarro (Thyroid Molecular Laboratory, Institute for Medical and Molecular Genetics, La Paz University Hospital, Autonomous University of Madrid, Madrid, Spain) for excellent technical work. The authors also acknowledge the computer resources provided by the Andalusia Bioinformatics Platform.

Funding.This work was funded by grants from the Ministerio de Economía y Competitividad, 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., and PI13/00593 and BFU2017-83588-P to B.R.G.). Human islets were procured through 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 article were 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 pancreatic islets, 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 wrote the manuscript. A.H., A.T., C.G.-G., J.S.W.R.H., L.H., and J.C.M. designed and performed human studies and transactivation assays. F.L. and D.B. provided human pancreatic islets. J.A. performed the human studies related to the P25R variant. B.R.G. is the guarantor of this work and, as such, had full access to all the

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data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Data Availability. The Microarray raw data on pancreatic islets overexpressing the murine or human PAX8 have been deposited in the Gene Expression Omnibus repository with the accession number GSE94846.

References

1. Baeyens L, Hindi S, Sorenson RL, German MS.b-Cell adaptation in pregnancy. Diabetes Obes Metab 2016;18(Suppl. 1):63–70

2. Rieck S, White P, Schug J, et al. The transcriptional response of the islet to pregnancy in mice. Mol Endocrinol 2009;23:1702–1712

3. Rieck S, Kaestner KH. Expansion of beta-cell mass in response to pregnancy.

Trends Endocrinol Metab 2010;21:151–158

4. Kim H, Toyofuku Y, Lynn FC, et al. Serotonin regulates pancreatic beta cell mass during pregnancy. Nat Med 2010;16:804–808

5. Mansouri A, Chowdhury K, Gruss P. Follicular cells of the thyroid gland require Pax8 gene function. Nat Genet 1998;19:87–90

6. Martin-Montalvo A, Lorenzo PI, López-Noriega L, Gauthier BR. Targeting pancreatic expressed PAX genes for the treatment of diabetes mellitus and pancreatic neuroendocrine tumors. Expert Opin Ther Targets 2017;21:77–89

7. Robson EJ, He SJ, Eccles MR. A PANorama of PAX genes in cancer and development. Nat Rev Cancer 2006;6:52–62

8. Lorenzo PI, Jimenez Moreno CM, Delgado I, et al. Immunohistochemical assessment of Pax8 expression during pancreatic islet development and in human neuroendocrine tumors. Histochem Cell Biol 2011;136:595–607

9. Moreno CM, Lorenzo PI, Delgado I, et al. Pax8 detection in well-differentiated pancreatic endocrine tumors: how reliable is it? Am J Surg Pathol 2011;35:1906–1908 10. Elbein SC, Das SK, Hallman DM, Hanis CL, Hasstedt SJ. Genome-wide linkage and admixture mapping of type 2 diabetes in African American families from the American Diabetes Association GENNID (Genetics of NIDDM) Study Cohort. Diabetes 2009;58:268–274

11. López-Noriega L, Cobo-Vuilleumier N, Narbona-Pérez AJ, et al. Levothyroxine enhances glucose clearance and blunts the onset of experimental type 1 diabetes mellitus in mice. Br J Pharmacol 2017;174:3795–3810

12. Lorenzo PI, Fuente-Martín E, Brun T, et al. PAX4 deﬁnes an expandableb-cell subpopulation in the adult pancreatic islet. Sci Rep 2015;5:15672

13. Jimenez-Moreno CM, Herrera-Gomez IG, Lopez-Noriega L, et al. A simple high efﬁciency intra-islet transduction protocol using lentiviral vectors. Curr Gene Ther 2015;15:436–446

14. Mellado-Gil JM, Fuente-Martín E, Lorenzo PI, et al. The type 2 diabetes- associated HMG20A gene is mandatory for islet beta cell functional maturity. Cell Death Dis 2018;9:279

15. Lu MC, Huang SS, Yan YH, Wang P. Use of the National Diabetes Data Group and the Carpenter-Coustan criteria for assessing gestational diabetes mellitus and risk of adverse pregnancy outcome. BMC Pregnancy Childbirth 2016;16:231

16. Carvalho A, Hermanns P, Rodrigues AL, et al. A new PAX8 mutation causing congenital hypothyroidism in three generations of a family is associated with abnormalities in the urogenital tract. Thyroid 2013;23:1074–1078

17. Moya CM, Zaballos MA, Garzon L, et al. TAZ/WWTR1 mediates the pulmonary effects of NKX2-1 mutations in Brain-Lung-Thyroid syndrome. J Clin Endocrinol Metab 2018;103:839–852

18. Abbassi-Ghanavati M, Greer LG, Cunningham FG. Pregnancy and laboratory studies: a reference table for clinicians. Obstet Gynecol 2009;114:1326–1331 19. Brun T, Hu He KH, Lupi R, et al. The diabetes-linked transcription factor Pax4 is expressed in human pancreatic islets and is activated by mitogens and GLP-1.

Hum Mol Genet 2008;17:478–489

20. Mittag J, Winterhager E, Bauer K, Grümmer R. Congenital hypothyroid female pax8-deﬁcient mice are infertile despite thyroid hormone replacement therapy.

Endocrinology 2007;148:719–725

21. Wistuba J, Mittag J, Luetjens CM, et al. Male congenital hypothyroid Pax8-/- mice are infertile despite adequate treatment with thyroid hormone. J Endocrinol 2007;192:99–109

22. Metzger BE, Gabbe SG, Persson B, et al.; International Association of Di- abetes and Pregnancy Study Groups Consensus Panel. International association of diabetes and pregnancy study groups recommendations on the diagnosis and classiﬁcation of hyperglycemia in pregnancy. Diabetes Care 2010;33:676– 682

23. Carpenter MW, Coustan DR. Criteria for screening tests for gestational diabetes. Am J Obstet Gynecol 1982;144:768–773

24. Bendl J, Stourac J, Salanda O, et al. PredictSNP: robust and accurate consensus classiﬁer for prediction of disease-related mutations. PLoS Comput Biol 2014;10:e1003440

25. Di Palma T, Nitsch R, Mascia A, Nitsch L, Di Lauro R, Zannini M. The paired domain-containing factor Pax8 and the homeodomain-containing factor TTF-1 directly interact and synergistically activate transcription. J Biol Chem 2003;278:

3395–3402

26. Scaglia L, Smith FE, Bonner-Weir S. Apoptosis contributes to the involution of beta cell mass in the post partum rat pancreas. Endocrinology 1995;136:5461– 5468

27. Di Palma T, Filippone MG, Pierantoni GM, Fusco A, Soddu S, Zannini M. Pax8 has a critical role in epithelial cell survival and proliferation. Cell Death Dis 2013;4:

e729

28. Yang D, Lai D, Huang X, et al. The defects in development and apoptosis of cardiomyocytes in mice lacking the transcriptional factor Pax-8. Int J Cardiol 2012;

154:43–51

29. Liu S, Wang X, Zou H, et al. Identiﬁcation and characterization of novel PAX8 mutations in Congenital Hypothyroidism(CH) in a Chinese population. Oncotarget 2017;8:8707–8716

30. Montanelli L, Tonacchera M. Genetics and phenomics of hypothyroidism and thyroid dys- and agenesis due to PAX8 and TTF1 mutations. Mol Cell Endocrinol 2010;322:64–71