Genetics, RWTH Aachen University, Aachen, Germany

Diabetes Care In Press, published online March 2, 2007

Digenic inheritance of HNF-1 and HNF-1 with MODY, polycystic thyroid and urogenital malformations

Running title: HNF-1 , HNF-1 , and digenic MODY

Received for publication 28 December 2006 and accepted in revised form 19 February 2007.

Beate Karges1, MD, Carsten Bergmann2, MD, Katrina Scholl1, MD, Eberhard Heinze1, MD, Franz Maximilian Rasche3, MD, Klaus Zerres2, MD, Klaus-Michael Debatin1, MD, Martin Wabitsch1, MD, Wolfram Karges4, MD

1Pediatric Endocrinology and Diabetes, University Children’s Hospital, University of Ulm, Ulm, Germany. 2Institute for Human Genetics, RWTH Aachen University, Aachen, Germany. 3Nephrology, University of Ulm, Ulm, Germany 4Endocrinology and Diabetes, RWTH Aachen University, Aachen, Germany.

Correspondence: Beate Karges, M.D. Pediatric Endocrinology and Diabetes University Children’s Hospital, University of Ulm Eythstrasse 24, D-89075 Ulm, Germany

E-mail: [email protected]

Abbreviations: MODY, maturity onset diabetes of the young; HNF, hepatocyte nuclear factor; HOMA, homeostasis model assessment; COUP-TF, chicken-ovalbumin-upstream-promoter transcription factor

Copyright American Diabetes Association, Inc., 2007 Heterozygous mutations of the POU Investigation A/homeodomain transcription factors Beta cell function was assessed after hepatocyte nuclear factor (HNF)- intravenous challenge with glucose (0.5 g/kg 1 andHNF-1 cause maturity onset bolus) and L-arginine (0.7g/min for 30 min). diabetes of the young (MODY) in humans In the index patient and her sisters, insulin (1). HNF-1 andHNF-1 act in a complex secretion was significantly impaired after IV network of transcription factors regulating glucose (maximum serum insulin 223.8, tissue-specific gene expression in the 124.1 and 145.6 pmol/l, respectively). In pancreas and other epithelial organs. contrast, there was a sustained insulin Patients with mutations of HNF-1β release after arginine infusion (maximum (MODY5) are characterized by urogenital serum insulin 543.1, 363.0, and 655.0 malformations, while extrapancreatic pmol/l, respectively). manifestations in patients with diabetes and Abdominal ultrasound in the index HNF-1 mutations (MODY3) are not well patient showed agenesis of the right kidney known (2). and a didelphic uterus with hypoplastic right uterine horn and hemiatresia of the cervix, History and examination which was confirmed by hysteroscopy. In After an occasional blood glucose both of her sisters, no abnormalities of the reading of 13.4 mmo/l, the diagnosis of urogenital system were identified by diabetes was established in a 13-year old ultrasound. In the index patient and the girl. She was asymptomatic except for mild younger sister, polycystic changes in both chronic lower abdominal discomfort. Her thyroid lobes with >50 cysts up to 4 mm in fasting glucose was 9.3 mmol/l, HbA1c was size were detected by high-resolution (12 8.5% (normal, 4.2-6.1%), and urine ketones MHz) ultrasound, and single thyroid cysts were negative. After brief insulin treatment, were identified in the father and the older she received glimepiride 0.5 mg/day, with a sister. No cystic or other lesions in the liver current HbA1c of 5.6%. were identified in any individual. Her father (Fig. 1), diagnosed with HNF-1β sequence analysis in the index diabetes at the age of 27 years, has received patient revealed a heterozygous genomic insulin since the age of 38 years. The missense variant (c.1006C>G) in exon 4, paternal grandmother had diabetes treated resulting in the substitution of a highly with glibenclamide until she died 20 years conserved residue (p.His336Asp) in the ago, aged 62 years. Two asymptomatic protein’s transactivation domain. This novel sisters of the index patient were diagnosed variant was detected in 1 of 400 with diabetes at the age of 14 years and 19 chromosomes in healthy individuals by years. Their fasting serum glucose was 7.3 denaturing high-performance liquid and 6.8 mmol/l, and HbA1c was 6.0 and chromatography. Sequencing identified 6.1%, respectively. 2 hours after a 75 g oral HNF-1β c.1006C>G in the younger sister glucose challenge, their glucose levels were and the father, but not in the older diabetic 10.1 and 13.1 mmol/l. sister. Thus, this mutation could not Fasting C-peptide was 299.7 pmol/l in exclusively account for the diabetes the index patient, and 599.4 and 566.1 phenotype in this family. pmol/l in her sisters; all had normal insulin We next analyzed HNF-1 and sensitivity (HOMA 2.0, 1.7 and 1.8, identified a heterozygous genomic mutation respectively) and normal BMI (<25 kg/m2). (c.526+1delGTAA) in the canonic splice Liver, kidney and thyroid function tests site of intron 2 in all diabetic individuals, including serum thyrotropin, free T4 and T3, but not in unaffected family members (Fig. creatinine, and other standard laboratory 1). This novel mutation is predicted to result parameters were normal in all sisters, and in aberrant HNF-1 splicing (3), with the serum islet cell autoantibodies and GAD65 introduction of a premature termination antibodies were negative. codon at amino acid position 194 and

deletion of the POU A and transactivation In several, but not all diabetic patients in domains. these kindred, a second mutation was To rule out further modifying gene identified in HNF-1β, leading to a non- effects, additional candidate genes conservative amino acid substitution in the putatively involved in the HNF highly conserved region of the transcriptional network were sequenced. In transactivation domain. This molecular the promoter of HNF-6, a sequential feature and the low allelic frequency suggest heterozygous single nucleotide that c.1006C>G is indeed a pathogenic polymorphism (c.1-400A>C, c.1-390C>A, HNF-1β variant. Strikingly, urogenital and c.1-385G>A) was identified in all diabetic polycystic thyroid changes but not metabolic patients, but also in the unaffected mother. characteristics were associated with the No sequence variation was detected in the mutant HNF-1β allele, while MODY index patient’s genomic DNA in the coding segregated with the HNF-1 variant. regions of HNF-4α, IPF1/PDX1, NeuroD In polarized epithelial cells, HNF-1 (causing MODY 1, 4, and 6, respectively), and HNF-1β cooperate in a network of nor in HNF-6, HNF-3β, COUP-TF I and transcriptional regulators including HNF-4 COUP-TF II. and HNF-3 , and both HNF-1 and β homo- and heterodimerize for DNA binding Conclusion via their N-terminal dimerization domains We have identified a novel heterozygous (6). It is conceivable that the extended HNF-1 splice site mutation that segregates MODY phenotype observed in these kindred with diabetes and impaired glucose- may result from the digenic inactivation of dependent insulin secretion, typical of HNF-1 and HNF-1β. In the kidney, MODY3 (4). In addition, polycystic thyroid, inactivation of HNF-1β inhibits the renal and genital abnormalities were found expression of the polycystic kidney disease to extend the clinical phenotype of MODY gene, Pkhd1 (7), and distinct functional in these kindred. In a recent report, renal characteristics of HNF-1β mutants lead to a agenesis has been described in two families spectrum of kidney malformations including with HNF-1 mutations, including a patient polycystic phenotypes (8). HNF-1 and with a different HNF-1 splice site variant HNF-1β interact with HNF-3β, a forkhead and a bicornute uterus (5). The association transcription factor expressed in early of HNF-1 mutants with a polycystic thyroid organogenesis and in the adult thyroid phenotype, however, has not been thyroid (9). The putative role of HNF-1 observed so far. Considering that HNF-1 transcription factors in thyroid disease mutations are the most common cause of involving differential transactivation MODY, extra-pancreatic manifestations ofHNF-3β or other target genes, however, seem overall rare in HNF-1 mutation must await experimental confirmation. carriers.

References

1. Fajans SS, Bell GI, Polonsky KS: Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young. N Engl J Med 345:971-980, 2001 2. Bellanne-Chantelot C, Chauveau D, Gautier JF, Dubois LaForgue D, Clauin S, Beaufils S, Wilhelm JM, Boitard C, Noel LH, Velho G, Timsit J: Clinical spectrum associated with hepatocyte nuclear factor-1beta mutations. Ann Intern Med 140:510- 517, 2004 3. Wang M, Marin A: Characterization and prediction of alternative splice sites. Gene 366:219-227, 2006 4. Pearson ER, Badmand MK, Lockwood CR, Clark PM, Ellard S, Bingham C, Hattersley AT: Contrasting diabetes phenotypes associated with hepatocyte nuclear factor -1 and -1 mutations. Diabetes Care 27:1102-1107, 2004 5. Malecki MT, Skupien J, Gorczynska-Kosiorz S, Klupa T, Nazim J, Moczulski DK, Sieradzki J: Renal malformations may be linked to mutations in the hepatocyte nuclear factor-1 alpha (MODY3) gene. Diabetes Care 28:2774-2776, 2005 6. Ryffel GU: Mutations of the human genes encoding the transcription factors of the hepatocyte nuclear factor (HNF)1 and HNF4 families: functional and pathological consequences. J Mol Endo 27:11-29, 2001 7. Hiesberger T, Bai Y, Shao X, McNally BT, Sinclair AM, Tian X, Somlo S, Igarashi P: Mutation of hepatocyte nuclear factor-1beta inhibits Pkhd1 gene expression and produces renal cysts in mice. J Clin Invest 113:814-825, 2004 8. Bohn S, Thomas HE, Turan G, Ellard S, Bingham C, Hattersley AT, Ryffel GU: Distinct molecular and morphogenetic properties of mutations in the human HNF1beta gene that lead to defective kidney development. J Am Soc Nephrol 14:2033-2041, 2003 9. de Felice M, di Lauro R: Thyroid development and its disorders: genetics and molecular mechanisms. Endocrine Reviews 25:722-746, 2004

Figure 1 Pedigree of family with maturity-onset diabetes of the young (MODY), polycystic thyroid and urogenital malformations. The clinical phenotype (filled symbols) is shown in relation to the HNF-1 (upper row) and HNF-1 allelic status (lower row) of each individual. not available.