Congenital Hyperinsulinism and Hypopituitarism Attributable to a Mutation in FOXA2

Congenital Hyperinsulinism and Hypopituitarism Attributable to a Mutation in FOXA2 Abstract Context Persistent hypoglycemia in the newborn period most commonly occurs as a result of hyperinsulinism. The phenotype of hypoketotic hypoglycemia can also result from pituitary hormone deficiencies, including growth hormone and adrenocorticotropic hormone deficiency. Forkhead box A2 (Foxa2) is a transcription factor shown in mouse models to influence insulin secretion by pancreatic β cells. In addition, Foxa2 is involved in regulation of pituitary development, and deletions of FOXA2 have been linked to panhypopituitarism. Objective To describe an infant with congenital hyperinsulinism and hypopituitarism as a result of a mutation in FOXA2 and to determine the functional impact of the identified mutation. Main Outcome Measure Difference in wild-type (WT) vs mutant Foxa2 transactivation of target genes that are critical for β cell function (ABCC8, KNCJ11, HADH) and pituitary development (GLI2, NKX2-2, SHH). Results Transactivation by mutant Foxa2 of all genes studied was substantially decreased compared with WT. Conclusions We report a mutation in FOXA2 leading to congenital hyperinsulinism and hypopituitarism and provide functional evidence of the molecular mechanism responsible for this phenotype. Congenital hyperinsulinism is the most common cause of persistent hypoglycemia in infants, but pituitary hormone deficiencies, including growth hormone and adrenocorticotropic hormone deficiency, can result in a similar phenotype of severe hypoketotic hypoglycemia. Diagnostic evaluation of neonates with persistent hypoglycemia must include an assessment for evidence of inappropriate insulin secretion, as well as pituitary hormone deficiency, to distinguish between these etiologies to determine the appropriate therapy. When both hypopituitarism and hyperinsulinism coexist, however, a unifying diagnosis should be sought. In this report, we describe an infant who presented with severe neonatal hypoglycemia as a result of both hypopituitarism and hyperinsulinism. Our finding of a mutation in forkhead box A2 (FOXA2) suggests a common etiology for this phenotype. Foxa2 is a member of the forkhead class of DNA-binding proteins that are involved in the development of multiple tissues, and global loss of function would likely be embryonic lethal, based on complete knockout studies (1). The role of Foxa2 on insulin secretion by mature β cells was demonstrated by the severe hypoglycemia phenotype of a mouse model with β cell-specific ablation of FOXA2 (2). Foxa2 has been shown to control the expression of several genes important in the regulation of insulin secretion, including ABCC8 and KCNJ11, which encode the Sur1 and Kir6.2 subunits of the adenosine triphosphate-sensitive potassium channel in pancreatic β cells (3). Inactivating mutations of these genes are the most common cause of congenital hyperinsulinism (4). Mutations in HADH, which encodes short-chain 3-hydroxyacyl-coenzyme A dehydrogenase, are a less common cause of congenital hyperinsulinism (5). Intron 1 of HADH has a highly conserved region that includes a Foxa2-binding site (6, 7), and previous studies have shown that its transactivation is regulated by Foxa2 (7, 8). In addition to its regulatory effects on β cell function, Foxa2 controls the expression of several genes involved in morphogenesis of the central nervous system, including Gli2, SHH, and Nkx2-2. Several cases of proximal 20p11 deletions, the cytogenetic location of FOXA2, have been described, with pituitary hormone deficiencies as a common feature (9–12). A recent report implicated FOXA2 in the pathogenesis of both hyperinsulinism and hypopituitarism (13). Here, we describe a child whose phenotype is consistent with hyperinsulinism and pituitary hormone deficiency, leading to severe hypoketotic hypoglycemia in the neonatal period, and demonstrate the molecular mechanism implicated in this complex phenotype. Subject and Methods Clinical data Clinical information was obtained by chart review. This single-patient case report did not require review by our Institutional Review Board. Genetic screening Genetic screening for known causes of congenital hyperinsulinism was carried out in a commercial laboratory. Written, informed consent for whole exome sequencing was obtained from the patient’s parents. Genomic DNA was extracted from peripheral blood, and targeted exons were captured and sequenced with 100 base pair paired-end reads. Mapping and analysis were based on the human genome build University of California Santa Cruz hg19 reference sequence. Sequencing data were processed using an in-house, custom-built bioinformatics pipeline. Coding exons and splice sites targeted were analyzed and reported. The following pathogenic variants are detectable using this methodology: single nucleotide variants, small deletions, and small insertions. Expression studies in 293T cells A pCMV6-XL5 expression vector containing full-length human FOXA2 cDNA was obtained from OriGene Technologies (Rockville, MD). Site-directed mutagenesis to create a point mutant for FOXA2 cDNA at 770 nucleotides G > T was performed using a QuickChange II Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA). Point mutation was confirmed by sequencing of the cloning plasmid (see Supplemental Materials for primers). Transfections of wild-type (WT) FOXA2 [pCMV6-XL5-FOXA2 WT (FOXA2 WT)] and mutant FOXA2 [pCMV6-XL5-FOXA2 mutant (FOXA2 mut)], as well as empty vector pCMV6-XL5 [pCMV6-XL5-empty vector (EV)] plasmids, into 293T cells were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Cells were harvested after 48 hours, and protein was extracted using radioimmunoprecipitation assay buffer. Western blot analysis from whole-cell extracts was performed using anti-Foxa2 antibody (Thermo Fisher, Rockford, IL). For the Western blot, loading protein quantity was standardized by equivalent β-actin bands [anti-β-actin antibody from Genscript (Piscataway, NJ)]. Cells were cotransfected with FOXA2 WT, FOXA2 mut, or EV (0.5 μg for each), 0.5 μg of one of six luciferase reporter plasmids (ABCC8, KCNJ11, HADH, GLI2, NKX2-2, or SHH), and 0.1 μg of a cytomegalovirus-β-galactosidase internal control plasmid (sources in Supplemental Materials). Cells were harvested 24 to 48 hours after transfection and assayed for luciferase activity (SwitchGear, Carlsbad, CA). All transfection assays were performed in triplicate. For the luciferase assay, cells were normalized to β-galactosidase activity. Data were analyzed by one-way analysis of variance. Results Clinical case summary An infant girl of Ashkenazi Jewish descent, born to unrelated parents, presented with hypoglycemia shortly after birth. She was born at 42 weeks gestation with a weight of 3.72 kg (appropriate for gestational age) to a 25-year-old nondiabetic mother. At ∼7 hours of life, she was found to be diaphoretic and hypothermic (36°C rectal). Plasma glucose was 16 mg/dL (0.89 mmol/L), confirmed on repeat by glucose meter. Hypoglycemia was managed with intravenous dextrose. While in the Neonatal Intensive Care Unit, she experienced recurrent hypoglycemia to 22 mg/dL (1.22 mmol/L) on day of life (DOL) 11. Results from a critical sample at the time of hypoglycemia were concerning for hypopituitarism (Table 1). Newborn screens obtained on DOL 3 and 6 were consistent with central hypothyroidism. Cortisol did not rise appropriately with a low-dose adrenocorticotropin stimulation test (Table 1), and hydrocortisone was started at 10 mg/m2/day on DOL 12. Levothyroxine 50 μg daily was started on DOL 13 and growth hormone replacement 0.35 mg/kg/week on DOL 14. Magnetic resonance imaging of the pituitary gland demonstrated small, shallow sella turcica with diminutive pituitary tissue, an ectopic posterior pituitary bright spot along the tuber cinereum, and nonvisualization/absence of the infundibulum (Fig. 1). Additional findings on the exam included coarse facial features, hypertelorism, thin upper lip, low-set ears, and widely spaced nipples. Table 1. Biochemical Evaluation at Baseline and During Hypoglycemia Test (Units, Reference Range)  Value  Initial work-up   T4 (newborn screen; μg/dL)      (a) Initial on DOL 3, (b) repeat on DOL 6  (a) 2.0, (b) <1.6   TSH (newborn screen; μIU/mL, <20)      (a) Initial on DOL 3, (b) repeat on DOL 6  (a) <2.91, (b) <2.91   Plasma glucose (mg/dL, 70–100)  22   Cortisol (μg/dL, 7–25)  0.9   Free T4 (ng/dL, 0.70–2.00)  0.28   TSH (mIU/mL, 0.30–5.00)  0.01   Insulin (μIU/mL, undetectable during hypoglycemia)  <1   Cortisol (μg/dL, >18.0)      (a) 30 Minutes, (b) 60 minutes after 1 μg ACTH  (a) 2.2, (b) 2.6  Critical sample at 1 mo of life, taken after diagnostic fast while receiving growth hormone and hydrocortisone; reference range given for sample measured during hypoglycemia (14)   Plasma glucose (mg/dL)  46   β-Hydroxybutyrate (mmol/L, ≥1.8)  0.80   Nonesterified free fatty acid (mmol/L, ≥1.7)  0.97   Insulin (μIU/mL, <2)  2.3   C-Peptide (ng/mL, <0.5 ng/mL)  0.483   Growth hormone (ng/mL, >7 ng/mL)  9.48 (2 Hours after growth hormone dose)   Ammonia (μmol/L, 9–33)  29   Lactate, plasma (mmol/L, 0.6–2.0)  1.4   Cortisol (μg/dL, 9–22)  <0.2 (8 Hours after hydrocortisone dose)   Change in plasma glucose in response to glucagon (mg/dL; positive response: ≥30 mg/dL)  62  Test (Units, Reference Range)  Value  Initial work-up   T4 (newborn screen; μg/dL)      (a) Initial on DOL 3, (b) repeat on DOL 6  (a) 2.0, (b) <1.6   TSH (newborn screen; μIU/mL, <20)      (a) Initial on DOL 3, (b) repeat on DOL 6  (a) <2.91, (b) <2.91   Plasma glucose (mg/dL, 70–100)  22   Cortisol (μg/dL, 7–25)  0.9   Free T4 (ng/dL, 0.70–2.00)  0.28   TSH (mIU/mL, 0.30–5.00)  0.01   Insulin (μIU/mL, undetectable during hypoglycemia)  <1   Cortisol (μg/dL, >18.0)      (a) 30 Minutes, (b) 60 minutes after 1 μg ACTH  (a) 2.2, (b) 2.6  Critical sample at 1 mo of life, taken after diagnostic fast while receiving growth hormone and hydrocortisone; reference range given for sample measured during hypoglycemia (14)   Plasma glucose (mg/dL)  46   β-Hydroxybutyrate (mmol/L, ≥1.8)  0.80   Nonesterified free fatty acid (mmol/L, ≥1.7)  0.97   Insulin (μIU/mL, <2)  2.3   C-Peptide (ng/mL, <0.5 ng/mL)  0.483   Growth hormone (ng/mL, >7 ng/mL)  9.48 (2 Hours after growth hormone dose)   Ammonia (μmol/L, 9–33)  29   Lactate, plasma (mmol/L, 0.6–2.0)  1.4   Cortisol (μg/dL, 9–22)  <0.2 (8 Hours after hydrocortisone dose)   Change in plasma glucose in response to glucagon (mg/dL; positive response: ≥30 mg/dL)  62  Abbreviations: ACTH, adrenocorticotropic hormone; TSH, thyroid-stimulating hormone. View Large Figure 1. View largeDownload slide Magnetic resonance imaging of the pituitary gland. T1 postcontrast images [(A) sagittal and (B) coronal] showing shallow sella turcica, hyperintensity typical of neurohypophysis located ectopically in the region of the tuber cinereum, and absence of the infundibulum. Lined arrows, pituitary bright spot; open arrows, typical location. Figure 1. View largeDownload slide Magnetic resonance imaging of the pituitary gland. T1 postcontrast images [(A) sagittal and (B) coronal] showing shallow sella turcica, hyperintensity typical of neurohypophysis located ectopically in the region of the tuber cinereum, and absence of the infundibulum. Lined arrows, pituitary bright spot; open arrows, typical location. Upon transfer to our institution on DOL 13, hypoglycemia persisted (<60 mg/dL, <3.33 mmol/L), despite pituitary hormone replacement, requiring a high-glucose infusion rate of 19 mg/kg/minute. On DOL 14, her hydrocortisone dose was increased to 20 mg/m2/day, and over the next couple of weeks, her glucose infusion requirement decreased gradually and was then weaned off. She continued to have hypoglycemia with prefeed plasma glucoses in the 50-mg/dL range, and a diagnostic fasting study was carried out at 1 month of life. Her growth hormone and hydrocortisone replacement were continued during the fast. Plasma glucose remained between 55 and 67 mg/dL (3.1 to 3.7 mmol/L) for the duration of the fast after the first 2 hours, falling to 46 mg/dL (2.6 mmol/L) after 12.5 hours. At the time of hypoglycemia, β-hydroxybutyrate and free fatty acid were suppressed. Glucagon stimulation test at the time of hypoglycemia resulted in a rise in plasma glucose of >30 mg/dL (1.67 mmol/L), consistent with excessive insulin action (Table 1) (14). She was started on diazoxide 5 mg/kg/day, divided twice daily. On the combination of diazoxide, growth hormone, and hydrocortisone, fasting tolerance improved to 14 hours with plasma glucose >70 mg/dL. Genetic screening Genetic screening for known causes of congenital hyperinsulinism detected no pathogenic sequence changes or deletion/duplication in ABCC8, GLUD1, HADH, HNF1A, HNF4A, INSR, KCNJ11, SLC16A1, and UCP2 and no pathogenic sequence change in GCK. Whole exome sequencing revealed a de novo c.770G > T, p.R257L FOXA2 variant of unknown importance. This variant was not observed in the National Heart, Lung, and Blood Institute Exome Sequencing Project Exome Variant Server (evs.gs.washington.edu/EVS), the 1000 Genomes database (1000genomes.org), or the Exome Aggregation Consortium Beta version database (http://exac.broadinstitute.org/). Functional analysis of the mutant Foxa2 The identified variant results in the nonconservative (positive-to-uncharged) substitution of an amino acid that is predicted to be highly evolutionarily conserved (conserved through frog) and that is located within a functional domain (forkhead transcription factor DNA-binding domain). This variant alters an amino acid that is located within the “wing 2,” a portion of the forkhead domain that is predicted to contact the minor groove of the DNA double helix (15). Because this change was anticipated to alter the function of the encoded protein, we sought to characterize the functional impact of the proband’s FOXA2 mutation. Site-directed mutagenesis was performed to recreate the mutation in a WT human FOXA2 expression plasmid. Mutant and WT human FOXA2 were expressed in 293T cells, and protein levels were evaluated by Western blot analysis. Expression levels were slightly lower for mutant compared with WT Foxa2 (Fig. 2A). Figure 2. View largeDownload slide Functional analysis of mutated Foxa2 protein. (A) Identification of mutant Foxa2 protein. WT and mutant (MT) forms of Homo sapiens Foxa2 protein expression levels in 293T cells were measured by Western blot analysis. Foxa2 protein is visualized at 48 kDa, and a β-actin protein is visualized at 42 kDa. (B) Functional analysis of mutant Foxa2. Mutant Foxa2 protein fails to activate ABCC8, KCNJ11, HADH, SHH, Gli2, and Nkx2-2. 293T cells were transfected with luciferase reporter plasmid, internal control plasmid, and expression plasmids (FOXA2 WT, FOXA2 MT, or EV). Values are expressed as means ± standard error of three independent transfections; P < 0.001 for all. Figure 2. View largeDownload slide Functional analysis of mutated Foxa2 protein. (A) Identification of mutant Foxa2 protein. WT and mutant (MT) forms of Homo sapiens Foxa2 protein expression levels in 293T cells were measured by Western blot analysis. Foxa2 protein is visualized at 48 kDa, and a β-actin protein is visualized at 42 kDa. (B) Functional analysis of mutant Foxa2. Mutant Foxa2 protein fails to activate ABCC8, KCNJ11, HADH, SHH, Gli2, and Nkx2-2. 293T cells were transfected with luciferase reporter plasmid, internal control plasmid, and expression plasmids (FOXA2 WT, FOXA2 MT, or EV). Values are expressed as means ± standard error of three independent transfections; P < 0.001 for all. To characterize the functional impact of the proband’s FOXA2 mutation, a luciferase reporter assay was developed to define the ability of mutant Foxa2 to transactivate target genes that are important for beta cell and pituitary development and function. Transactivation of all genes studied by mutant Foxa2 was substantially decreased compared with WT Foxa2 (ABCC8: 30.8% decrease; KCNJ11: 27.0% decrease; HADH: 25.7% decrease; SHH: 67.3% decrease; Gli2: 62.9% decrease; and NKX2-2: 52.1% decrease; P < 0.001 for all; n = 3 for each; Fig. 2B). Discussion Our patient presented with hypoketotic hypoglycemia, which was initially attributed to pituitary hormone deficiencies. Despite correction of these deficiencies, hypoketotic hypoglycemia persisted, and her additional diagnosis of congenital hyperinsulinism became evident. Although in the neonatal period, hypopituitarism is a known mimicker of hyperinsulinism as a result of the failure to activate ketogenesis appropriately at the time of hypoglycemia, hypopituitarism and hyperinsulinism are often thought of as distinct entities. Here, we report a missense mutation (c.770G > T, p.R257L) in FOXA2 in an infant with neonatal hypoketotic hypoglycemia that appears to link hyperinsulinism and hypopituitarism. This mutation occurred de novo and reduces expression of genes that encode key components of the insulin secretory pathway, as well as genes that impact pituitary gland development, supportive of a causal relationship between the identified mutation and our patient’s phenotype. The role of Foxa2 in pituitary gland development is suggested by a recent case report by Giri et al. (13), as well as reports of proximal 20p deletions with associated panhypopituitarism, suspected to occur as a result of cross-regulatory interactions between the SHH/Gli signaling pathway and FOXA2 and NKx2.2 genes (9–11). Additional studies highlight the impact of mutations of the downstream transcription factor Gli2, both in mice and in humans, with resultant absence of the pituitary or combined pituitary hormone deficiencies, respectively (16, 17). As demonstrated by our functional studies, our patient’s mutation appears to decrease substantially expression of SHH, Gli2, and NKX2-2, consistent with her abnormal pituitary findings. Our results also suggest that our patient’s hyperinsulinism is the result of dysregulated insulin secretion as a result of reduced expression of ABCC8 and KCNJ11, but her responsiveness to diazoxide, an activator of the adenosine triphosphate-sensitive potassium channel, suggests only a partial, not complete, reduction. Our finding of reduced expression of HADH, a target of Foxa2, suggests an additional mechanism of hyperinsulinism. Of note, our patient did not have hyperammonemia. Unlike GDH hyperinsulinism, in which abnormal GDH activity in the kidney results in hyperammonemia, in hyperinsulinism as a result of mutations in HADH, GDH activity in the kidney is unaffected, and ammonia is not elevated. Mutations in HADH lead to both fasting and protein-induced hypoglycemia as a result of abnormal regulation of glutamate dehydrogenase activity (18, 19). A protein tolerance test was not completed for our patient before initiation of diazoxide but would be expected to elicit a hypoglycemic response to protein load. It is currently unknown whether this patient, like other patients with congenital hyperinsulinism, will demonstrate improvement in glycemic control with age (20). At 2 years and 2 months of age, she remains on a low dose of diazoxide (4.8 mg/kg/day) and growth hormone (0.1 mg/kg/week) with robust linear growth (length >90th percentile), as well as a standard physiological dose of hydrocortisone (10 mg/m2/day) without substantial hypoglycemia. Conclusion Our study reports a mutation in FOXA2 and elucidates the molecular mechanisms of hypopituitarism and hyperinsulinism through functional analysis of the mutation’s impact on target genes expressed in beta cells and the pituitary gland. Our patient’s mutation demonstrates the important role that Foxa2 plays in both pancreatic and pituitary development/function and highlights the importance of thorough diagnostic evaluations and appropriate treatment of neonatal hypoglycemia. Abbreviations: DOL day of life EV pCMV6-XL5-empty vector Foxa2 forkhead box A2 FOXA2 mut pCMV6-XL5-forkhead box A2 mutant FOXA2 WT pCMV6-XL5-forkhead box A2 wild-type WT wild-type. Acknowledgments We acknowledge the patient described in this study and her family, as well as the hyperinsulinism team at the Children’s Hospital of Philadelphia Hyperinsulinism Center. Financial Support: This work was supported by National Institutes of Health Grants 1R01DK098517 (to D.D.D.L.) and T32 DK07314 (to M.E.V.), as well as a grant from the Endocrine Fellows Foundation (to M.E.V.). M.E.V. is a fellow of the Center for Healthcare Improvement and Patient Safety and the Leonard Davis Institute of Health Economics at the University of Pennsylvania. Author Contributions: M.E.V. collected clinical data and wrote the first draft of the manuscript. J.C. performed the expression studies and functional analysis. B.K. and S.B. performed analysis of whole exome sequencing results. D.L. and C.A. contributed to collection of clinical data and edited the manuscript. D.D.D.L. directed the study and edited the manuscript. Disclosure Summary: The authors have nothing to disclose. References 1. Ang SL, Rossant J. HNF-3 beta is essential for node and notochord formation in mouse development. Cell . 1994; 78( 4): 561– 574. Google Scholar CrossRef Search ADS PubMed  2. Gao N, Le Lay J, Qin W, Doliba N, Schug J, Fox AJ, Smirnova O, Matschinsky FM, Kaestner KH. Foxa1 and Foxa2 maintain the metabolic and secretory features of the mature β-cell. Mol Endocrinol . 2010; 24( 8): 1594– 1604. Google Scholar CrossRef Search ADS PubMed  3. Heddad Masson M, Poisson C, Guérardel A, Mamin A, Philippe J, Gosmain Y. Foxa1 and Foxa2 regulate α-cell differentiation, glucagon biosynthesis, and secretion. Endocrinology . 2014; 155( 10): 3781– 3792. Google Scholar CrossRef Search ADS PubMed  4. Stanley CA. Perspective on the genetics and diagnosis of congenital hyperinsulinism disorders. J Clin Endocrinol Metab . 2016; 101( 3): 815– 826. Google Scholar CrossRef Search ADS PubMed  5. Kapoor RR, Heslegrave A, Hussain K. Congenital hyperinsulinism due to mutations in HNF4A and HADH. Rev Endocr Metab Disord . 2010; 11( 3): 185– 191. Google Scholar CrossRef Search ADS PubMed  6. Overdier DG, Porcella A, Costa RH. The DNA-binding specificity of the hepatocyte nuclear factor 3/forkhead domain is influenced by amino-acid residues adjacent to the recognition helix. Mol Cell Biol . 1994; 14( 4): 2755– 2766. Google Scholar CrossRef Search ADS PubMed  7. Lantz KA, Vatamaniuk MZ, Brestelli JE, Friedman JR, Matschinsky FM, Kaestner KH. Foxa2 regulates multiple pathways of insulin secretion. 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Biomarkers of insulin for the diagnosis of hyperinsulinemic hypoglycemia in infants and children. J Pediatr . 2016; 168: 212– 219. Google Scholar CrossRef Search ADS PubMed  15. Clark KL, Halay ED, Lai E, Burley SK. Co-crystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5. Nature . 1993; 364( 6436): 412– 420. Google Scholar CrossRef Search ADS PubMed  16. Park HL, Bai C, Platt KA, Matise MP, Beeghly A, Hui CC, Nakashima M, Joyner AL. Mouse Gli1 mutants are viable but have defects in SHH signaling in combination with a Gli2 mutation. Development . 2000; 127( 8): 1593– 1605. Google Scholar PubMed  17. França MM, Jorge AAL, Carvalho LRS, Costalonga EF, Vasques GA, Leite CC, Mendonca BB, Arnhold IJP. Novel heterozygous nonsense GLI2 mutations in patients with hypopituitarism and ectopic posterior pituitary lobe without holoprosencephaly. J Clin Endocrinol Metab . 2010; 95( 11): E384– E391. Google Scholar CrossRef Search ADS PubMed  18. Kapoor RR, James C, Flanagan SE, Ellard S, Eaton S, Hussain K. 3-Hydroxyacyl-coenzyme A dehydrogenase deficiency and hyperinsulinemic hypoglycemia: characterization of a novel mutation and severe dietary protein sensitivity. J Clin Endocrinol Metab . 2009; 94( 7): 2221– 2225. Google Scholar CrossRef Search ADS PubMed  19. Li C, Chen P, Palladino A, Narayan S, Russell LK, Sayed S, Xiong G, Chen J, Stokes D, Butt YM, Jones PM, Collins HW, Cohen NA, Cohen AS, Nissim I, Smith TJ, Strauss AW, Matschinsky FM, Bennett MJ, Stanley CA. Mechanism of hyperinsulinism in short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency involves activation of glutamate dehydrogenase. J Biol Chem . 2010; 285( 41): 31806– 31818. Google Scholar CrossRef Search ADS PubMed  20. Salomon-Estebanez M, Flanagan SE, Ellard S, Rigby L, Bowden L, Mohamed Z, Nicholson J, Skae M, Hall C, Craigie R, Padidela R, Murphy N, Randell T, Cosgrove KE, Dunne MJ, Banerjee I. Conservatively treated congenital hyperinsulinism (CHI) due to K-ATP channel gene mutations: reducing severity over time. Orphanet J Rare Dis . 2016; 11( 1): 163. Google Scholar CrossRef Search ADS PubMed  Copyright © 2018 Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Clinical Endocrinology and Metabolism Oxford University Press

Congenital Hyperinsulinism and Hypopituitarism Attributable to a Mutation in FOXA2

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Endocrine Society
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Copyright © 2018 Endocrine Society
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0021-972X
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10.1210/jc.2017-02157
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Abstract

Abstract Context Persistent hypoglycemia in the newborn period most commonly occurs as a result of hyperinsulinism. The phenotype of hypoketotic hypoglycemia can also result from pituitary hormone deficiencies, including growth hormone and adrenocorticotropic hormone deficiency. Forkhead box A2 (Foxa2) is a transcription factor shown in mouse models to influence insulin secretion by pancreatic β cells. In addition, Foxa2 is involved in regulation of pituitary development, and deletions of FOXA2 have been linked to panhypopituitarism. Objective To describe an infant with congenital hyperinsulinism and hypopituitarism as a result of a mutation in FOXA2 and to determine the functional impact of the identified mutation. Main Outcome Measure Difference in wild-type (WT) vs mutant Foxa2 transactivation of target genes that are critical for β cell function (ABCC8, KNCJ11, HADH) and pituitary development (GLI2, NKX2-2, SHH). Results Transactivation by mutant Foxa2 of all genes studied was substantially decreased compared with WT. Conclusions We report a mutation in FOXA2 leading to congenital hyperinsulinism and hypopituitarism and provide functional evidence of the molecular mechanism responsible for this phenotype. Congenital hyperinsulinism is the most common cause of persistent hypoglycemia in infants, but pituitary hormone deficiencies, including growth hormone and adrenocorticotropic hormone deficiency, can result in a similar phenotype of severe hypoketotic hypoglycemia. Diagnostic evaluation of neonates with persistent hypoglycemia must include an assessment for evidence of inappropriate insulin secretion, as well as pituitary hormone deficiency, to distinguish between these etiologies to determine the appropriate therapy. When both hypopituitarism and hyperinsulinism coexist, however, a unifying diagnosis should be sought. In this report, we describe an infant who presented with severe neonatal hypoglycemia as a result of both hypopituitarism and hyperinsulinism. Our finding of a mutation in forkhead box A2 (FOXA2) suggests a common etiology for this phenotype. Foxa2 is a member of the forkhead class of DNA-binding proteins that are involved in the development of multiple tissues, and global loss of function would likely be embryonic lethal, based on complete knockout studies (1). The role of Foxa2 on insulin secretion by mature β cells was demonstrated by the severe hypoglycemia phenotype of a mouse model with β cell-specific ablation of FOXA2 (2). Foxa2 has been shown to control the expression of several genes important in the regulation of insulin secretion, including ABCC8 and KCNJ11, which encode the Sur1 and Kir6.2 subunits of the adenosine triphosphate-sensitive potassium channel in pancreatic β cells (3). Inactivating mutations of these genes are the most common cause of congenital hyperinsulinism (4). Mutations in HADH, which encodes short-chain 3-hydroxyacyl-coenzyme A dehydrogenase, are a less common cause of congenital hyperinsulinism (5). Intron 1 of HADH has a highly conserved region that includes a Foxa2-binding site (6, 7), and previous studies have shown that its transactivation is regulated by Foxa2 (7, 8). In addition to its regulatory effects on β cell function, Foxa2 controls the expression of several genes involved in morphogenesis of the central nervous system, including Gli2, SHH, and Nkx2-2. Several cases of proximal 20p11 deletions, the cytogenetic location of FOXA2, have been described, with pituitary hormone deficiencies as a common feature (9–12). A recent report implicated FOXA2 in the pathogenesis of both hyperinsulinism and hypopituitarism (13). Here, we describe a child whose phenotype is consistent with hyperinsulinism and pituitary hormone deficiency, leading to severe hypoketotic hypoglycemia in the neonatal period, and demonstrate the molecular mechanism implicated in this complex phenotype. Subject and Methods Clinical data Clinical information was obtained by chart review. This single-patient case report did not require review by our Institutional Review Board. Genetic screening Genetic screening for known causes of congenital hyperinsulinism was carried out in a commercial laboratory. Written, informed consent for whole exome sequencing was obtained from the patient’s parents. Genomic DNA was extracted from peripheral blood, and targeted exons were captured and sequenced with 100 base pair paired-end reads. Mapping and analysis were based on the human genome build University of California Santa Cruz hg19 reference sequence. Sequencing data were processed using an in-house, custom-built bioinformatics pipeline. Coding exons and splice sites targeted were analyzed and reported. The following pathogenic variants are detectable using this methodology: single nucleotide variants, small deletions, and small insertions. Expression studies in 293T cells A pCMV6-XL5 expression vector containing full-length human FOXA2 cDNA was obtained from OriGene Technologies (Rockville, MD). Site-directed mutagenesis to create a point mutant for FOXA2 cDNA at 770 nucleotides G > T was performed using a QuickChange II Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA). Point mutation was confirmed by sequencing of the cloning plasmid (see Supplemental Materials for primers). Transfections of wild-type (WT) FOXA2 [pCMV6-XL5-FOXA2 WT (FOXA2 WT)] and mutant FOXA2 [pCMV6-XL5-FOXA2 mutant (FOXA2 mut)], as well as empty vector pCMV6-XL5 [pCMV6-XL5-empty vector (EV)] plasmids, into 293T cells were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Cells were harvested after 48 hours, and protein was extracted using radioimmunoprecipitation assay buffer. Western blot analysis from whole-cell extracts was performed using anti-Foxa2 antibody (Thermo Fisher, Rockford, IL). For the Western blot, loading protein quantity was standardized by equivalent β-actin bands [anti-β-actin antibody from Genscript (Piscataway, NJ)]. Cells were cotransfected with FOXA2 WT, FOXA2 mut, or EV (0.5 μg for each), 0.5 μg of one of six luciferase reporter plasmids (ABCC8, KCNJ11, HADH, GLI2, NKX2-2, or SHH), and 0.1 μg of a cytomegalovirus-β-galactosidase internal control plasmid (sources in Supplemental Materials). Cells were harvested 24 to 48 hours after transfection and assayed for luciferase activity (SwitchGear, Carlsbad, CA). All transfection assays were performed in triplicate. For the luciferase assay, cells were normalized to β-galactosidase activity. Data were analyzed by one-way analysis of variance. Results Clinical case summary An infant girl of Ashkenazi Jewish descent, born to unrelated parents, presented with hypoglycemia shortly after birth. She was born at 42 weeks gestation with a weight of 3.72 kg (appropriate for gestational age) to a 25-year-old nondiabetic mother. At ∼7 hours of life, she was found to be diaphoretic and hypothermic (36°C rectal). Plasma glucose was 16 mg/dL (0.89 mmol/L), confirmed on repeat by glucose meter. Hypoglycemia was managed with intravenous dextrose. While in the Neonatal Intensive Care Unit, she experienced recurrent hypoglycemia to 22 mg/dL (1.22 mmol/L) on day of life (DOL) 11. Results from a critical sample at the time of hypoglycemia were concerning for hypopituitarism (Table 1). Newborn screens obtained on DOL 3 and 6 were consistent with central hypothyroidism. Cortisol did not rise appropriately with a low-dose adrenocorticotropin stimulation test (Table 1), and hydrocortisone was started at 10 mg/m2/day on DOL 12. Levothyroxine 50 μg daily was started on DOL 13 and growth hormone replacement 0.35 mg/kg/week on DOL 14. Magnetic resonance imaging of the pituitary gland demonstrated small, shallow sella turcica with diminutive pituitary tissue, an ectopic posterior pituitary bright spot along the tuber cinereum, and nonvisualization/absence of the infundibulum (Fig. 1). Additional findings on the exam included coarse facial features, hypertelorism, thin upper lip, low-set ears, and widely spaced nipples. Table 1. Biochemical Evaluation at Baseline and During Hypoglycemia Test (Units, Reference Range)  Value  Initial work-up   T4 (newborn screen; μg/dL)      (a) Initial on DOL 3, (b) repeat on DOL 6  (a) 2.0, (b) <1.6   TSH (newborn screen; μIU/mL, <20)      (a) Initial on DOL 3, (b) repeat on DOL 6  (a) <2.91, (b) <2.91   Plasma glucose (mg/dL, 70–100)  22   Cortisol (μg/dL, 7–25)  0.9   Free T4 (ng/dL, 0.70–2.00)  0.28   TSH (mIU/mL, 0.30–5.00)  0.01   Insulin (μIU/mL, undetectable during hypoglycemia)  <1   Cortisol (μg/dL, >18.0)      (a) 30 Minutes, (b) 60 minutes after 1 μg ACTH  (a) 2.2, (b) 2.6  Critical sample at 1 mo of life, taken after diagnostic fast while receiving growth hormone and hydrocortisone; reference range given for sample measured during hypoglycemia (14)   Plasma glucose (mg/dL)  46   β-Hydroxybutyrate (mmol/L, ≥1.8)  0.80   Nonesterified free fatty acid (mmol/L, ≥1.7)  0.97   Insulin (μIU/mL, <2)  2.3   C-Peptide (ng/mL, <0.5 ng/mL)  0.483   Growth hormone (ng/mL, >7 ng/mL)  9.48 (2 Hours after growth hormone dose)   Ammonia (μmol/L, 9–33)  29   Lactate, plasma (mmol/L, 0.6–2.0)  1.4   Cortisol (μg/dL, 9–22)  <0.2 (8 Hours after hydrocortisone dose)   Change in plasma glucose in response to glucagon (mg/dL; positive response: ≥30 mg/dL)  62  Test (Units, Reference Range)  Value  Initial work-up   T4 (newborn screen; μg/dL)      (a) Initial on DOL 3, (b) repeat on DOL 6  (a) 2.0, (b) <1.6   TSH (newborn screen; μIU/mL, <20)      (a) Initial on DOL 3, (b) repeat on DOL 6  (a) <2.91, (b) <2.91   Plasma glucose (mg/dL, 70–100)  22   Cortisol (μg/dL, 7–25)  0.9   Free T4 (ng/dL, 0.70–2.00)  0.28   TSH (mIU/mL, 0.30–5.00)  0.01   Insulin (μIU/mL, undetectable during hypoglycemia)  <1   Cortisol (μg/dL, >18.0)      (a) 30 Minutes, (b) 60 minutes after 1 μg ACTH  (a) 2.2, (b) 2.6  Critical sample at 1 mo of life, taken after diagnostic fast while receiving growth hormone and hydrocortisone; reference range given for sample measured during hypoglycemia (14)   Plasma glucose (mg/dL)  46   β-Hydroxybutyrate (mmol/L, ≥1.8)  0.80   Nonesterified free fatty acid (mmol/L, ≥1.7)  0.97   Insulin (μIU/mL, <2)  2.3   C-Peptide (ng/mL, <0.5 ng/mL)  0.483   Growth hormone (ng/mL, >7 ng/mL)  9.48 (2 Hours after growth hormone dose)   Ammonia (μmol/L, 9–33)  29   Lactate, plasma (mmol/L, 0.6–2.0)  1.4   Cortisol (μg/dL, 9–22)  <0.2 (8 Hours after hydrocortisone dose)   Change in plasma glucose in response to glucagon (mg/dL; positive response: ≥30 mg/dL)  62  Abbreviations: ACTH, adrenocorticotropic hormone; TSH, thyroid-stimulating hormone. View Large Figure 1. View largeDownload slide Magnetic resonance imaging of the pituitary gland. T1 postcontrast images [(A) sagittal and (B) coronal] showing shallow sella turcica, hyperintensity typical of neurohypophysis located ectopically in the region of the tuber cinereum, and absence of the infundibulum. Lined arrows, pituitary bright spot; open arrows, typical location. Figure 1. View largeDownload slide Magnetic resonance imaging of the pituitary gland. T1 postcontrast images [(A) sagittal and (B) coronal] showing shallow sella turcica, hyperintensity typical of neurohypophysis located ectopically in the region of the tuber cinereum, and absence of the infundibulum. Lined arrows, pituitary bright spot; open arrows, typical location. Upon transfer to our institution on DOL 13, hypoglycemia persisted (<60 mg/dL, <3.33 mmol/L), despite pituitary hormone replacement, requiring a high-glucose infusion rate of 19 mg/kg/minute. On DOL 14, her hydrocortisone dose was increased to 20 mg/m2/day, and over the next couple of weeks, her glucose infusion requirement decreased gradually and was then weaned off. She continued to have hypoglycemia with prefeed plasma glucoses in the 50-mg/dL range, and a diagnostic fasting study was carried out at 1 month of life. Her growth hormone and hydrocortisone replacement were continued during the fast. Plasma glucose remained between 55 and 67 mg/dL (3.1 to 3.7 mmol/L) for the duration of the fast after the first 2 hours, falling to 46 mg/dL (2.6 mmol/L) after 12.5 hours. At the time of hypoglycemia, β-hydroxybutyrate and free fatty acid were suppressed. Glucagon stimulation test at the time of hypoglycemia resulted in a rise in plasma glucose of >30 mg/dL (1.67 mmol/L), consistent with excessive insulin action (Table 1) (14). She was started on diazoxide 5 mg/kg/day, divided twice daily. On the combination of diazoxide, growth hormone, and hydrocortisone, fasting tolerance improved to 14 hours with plasma glucose >70 mg/dL. Genetic screening Genetic screening for known causes of congenital hyperinsulinism detected no pathogenic sequence changes or deletion/duplication in ABCC8, GLUD1, HADH, HNF1A, HNF4A, INSR, KCNJ11, SLC16A1, and UCP2 and no pathogenic sequence change in GCK. Whole exome sequencing revealed a de novo c.770G > T, p.R257L FOXA2 variant of unknown importance. This variant was not observed in the National Heart, Lung, and Blood Institute Exome Sequencing Project Exome Variant Server (evs.gs.washington.edu/EVS), the 1000 Genomes database (1000genomes.org), or the Exome Aggregation Consortium Beta version database (http://exac.broadinstitute.org/). Functional analysis of the mutant Foxa2 The identified variant results in the nonconservative (positive-to-uncharged) substitution of an amino acid that is predicted to be highly evolutionarily conserved (conserved through frog) and that is located within a functional domain (forkhead transcription factor DNA-binding domain). This variant alters an amino acid that is located within the “wing 2,” a portion of the forkhead domain that is predicted to contact the minor groove of the DNA double helix (15). Because this change was anticipated to alter the function of the encoded protein, we sought to characterize the functional impact of the proband’s FOXA2 mutation. Site-directed mutagenesis was performed to recreate the mutation in a WT human FOXA2 expression plasmid. Mutant and WT human FOXA2 were expressed in 293T cells, and protein levels were evaluated by Western blot analysis. Expression levels were slightly lower for mutant compared with WT Foxa2 (Fig. 2A). Figure 2. View largeDownload slide Functional analysis of mutated Foxa2 protein. (A) Identification of mutant Foxa2 protein. WT and mutant (MT) forms of Homo sapiens Foxa2 protein expression levels in 293T cells were measured by Western blot analysis. Foxa2 protein is visualized at 48 kDa, and a β-actin protein is visualized at 42 kDa. (B) Functional analysis of mutant Foxa2. Mutant Foxa2 protein fails to activate ABCC8, KCNJ11, HADH, SHH, Gli2, and Nkx2-2. 293T cells were transfected with luciferase reporter plasmid, internal control plasmid, and expression plasmids (FOXA2 WT, FOXA2 MT, or EV). Values are expressed as means ± standard error of three independent transfections; P < 0.001 for all. Figure 2. View largeDownload slide Functional analysis of mutated Foxa2 protein. (A) Identification of mutant Foxa2 protein. WT and mutant (MT) forms of Homo sapiens Foxa2 protein expression levels in 293T cells were measured by Western blot analysis. Foxa2 protein is visualized at 48 kDa, and a β-actin protein is visualized at 42 kDa. (B) Functional analysis of mutant Foxa2. Mutant Foxa2 protein fails to activate ABCC8, KCNJ11, HADH, SHH, Gli2, and Nkx2-2. 293T cells were transfected with luciferase reporter plasmid, internal control plasmid, and expression plasmids (FOXA2 WT, FOXA2 MT, or EV). Values are expressed as means ± standard error of three independent transfections; P < 0.001 for all. To characterize the functional impact of the proband’s FOXA2 mutation, a luciferase reporter assay was developed to define the ability of mutant Foxa2 to transactivate target genes that are important for beta cell and pituitary development and function. Transactivation of all genes studied by mutant Foxa2 was substantially decreased compared with WT Foxa2 (ABCC8: 30.8% decrease; KCNJ11: 27.0% decrease; HADH: 25.7% decrease; SHH: 67.3% decrease; Gli2: 62.9% decrease; and NKX2-2: 52.1% decrease; P < 0.001 for all; n = 3 for each; Fig. 2B). Discussion Our patient presented with hypoketotic hypoglycemia, which was initially attributed to pituitary hormone deficiencies. Despite correction of these deficiencies, hypoketotic hypoglycemia persisted, and her additional diagnosis of congenital hyperinsulinism became evident. Although in the neonatal period, hypopituitarism is a known mimicker of hyperinsulinism as a result of the failure to activate ketogenesis appropriately at the time of hypoglycemia, hypopituitarism and hyperinsulinism are often thought of as distinct entities. Here, we report a missense mutation (c.770G > T, p.R257L) in FOXA2 in an infant with neonatal hypoketotic hypoglycemia that appears to link hyperinsulinism and hypopituitarism. This mutation occurred de novo and reduces expression of genes that encode key components of the insulin secretory pathway, as well as genes that impact pituitary gland development, supportive of a causal relationship between the identified mutation and our patient’s phenotype. The role of Foxa2 in pituitary gland development is suggested by a recent case report by Giri et al. (13), as well as reports of proximal 20p deletions with associated panhypopituitarism, suspected to occur as a result of cross-regulatory interactions between the SHH/Gli signaling pathway and FOXA2 and NKx2.2 genes (9–11). Additional studies highlight the impact of mutations of the downstream transcription factor Gli2, both in mice and in humans, with resultant absence of the pituitary or combined pituitary hormone deficiencies, respectively (16, 17). As demonstrated by our functional studies, our patient’s mutation appears to decrease substantially expression of SHH, Gli2, and NKX2-2, consistent with her abnormal pituitary findings. Our results also suggest that our patient’s hyperinsulinism is the result of dysregulated insulin secretion as a result of reduced expression of ABCC8 and KCNJ11, but her responsiveness to diazoxide, an activator of the adenosine triphosphate-sensitive potassium channel, suggests only a partial, not complete, reduction. Our finding of reduced expression of HADH, a target of Foxa2, suggests an additional mechanism of hyperinsulinism. Of note, our patient did not have hyperammonemia. Unlike GDH hyperinsulinism, in which abnormal GDH activity in the kidney results in hyperammonemia, in hyperinsulinism as a result of mutations in HADH, GDH activity in the kidney is unaffected, and ammonia is not elevated. Mutations in HADH lead to both fasting and protein-induced hypoglycemia as a result of abnormal regulation of glutamate dehydrogenase activity (18, 19). A protein tolerance test was not completed for our patient before initiation of diazoxide but would be expected to elicit a hypoglycemic response to protein load. It is currently unknown whether this patient, like other patients with congenital hyperinsulinism, will demonstrate improvement in glycemic control with age (20). At 2 years and 2 months of age, she remains on a low dose of diazoxide (4.8 mg/kg/day) and growth hormone (0.1 mg/kg/week) with robust linear growth (length >90th percentile), as well as a standard physiological dose of hydrocortisone (10 mg/m2/day) without substantial hypoglycemia. Conclusion Our study reports a mutation in FOXA2 and elucidates the molecular mechanisms of hypopituitarism and hyperinsulinism through functional analysis of the mutation’s impact on target genes expressed in beta cells and the pituitary gland. Our patient’s mutation demonstrates the important role that Foxa2 plays in both pancreatic and pituitary development/function and highlights the importance of thorough diagnostic evaluations and appropriate treatment of neonatal hypoglycemia. Abbreviations: DOL day of life EV pCMV6-XL5-empty vector Foxa2 forkhead box A2 FOXA2 mut pCMV6-XL5-forkhead box A2 mutant FOXA2 WT pCMV6-XL5-forkhead box A2 wild-type WT wild-type. Acknowledgments We acknowledge the patient described in this study and her family, as well as the hyperinsulinism team at the Children’s Hospital of Philadelphia Hyperinsulinism Center. Financial Support: This work was supported by National Institutes of Health Grants 1R01DK098517 (to D.D.D.L.) and T32 DK07314 (to M.E.V.), as well as a grant from the Endocrine Fellows Foundation (to M.E.V.). M.E.V. is a fellow of the Center for Healthcare Improvement and Patient Safety and the Leonard Davis Institute of Health Economics at the University of Pennsylvania. Author Contributions: M.E.V. collected clinical data and wrote the first draft of the manuscript. J.C. performed the expression studies and functional analysis. B.K. and S.B. performed analysis of whole exome sequencing results. D.L. and C.A. contributed to collection of clinical data and edited the manuscript. D.D.D.L. directed the study and edited the manuscript. Disclosure Summary: The authors have nothing to disclose. References 1. Ang SL, Rossant J. HNF-3 beta is essential for node and notochord formation in mouse development. Cell . 1994; 78( 4): 561– 574. Google Scholar CrossRef Search ADS PubMed  2. Gao N, Le Lay J, Qin W, Doliba N, Schug J, Fox AJ, Smirnova O, Matschinsky FM, Kaestner KH. Foxa1 and Foxa2 maintain the metabolic and secretory features of the mature β-cell. Mol Endocrinol . 2010; 24( 8): 1594– 1604. Google Scholar CrossRef Search ADS PubMed  3. Heddad Masson M, Poisson C, Guérardel A, Mamin A, Philippe J, Gosmain Y. 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Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Mar 1, 2018

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