Digenic inheritance of HNF-1 and HNF-1 with MODY, polycystic thyroid and urogenital malformations. Running title: HNF-1, HNF-1, and digenic MODY

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1 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 Beate Karges 1, MD, Carsten Bergmann 2, MD, Katrina Scholl 1, MD, Eberhard Heinze 1, MD, Franz Maximilian Rasche 3, MD, Klaus Zerres 2, MD, Klaus-Michael Debatin 1, MD, Martin Wabitsch 1, MD, Wolfram Karges 4, MD 1 Pediatric Endocrinology and Diabetes, University Children s Hospital, University of Ulm, Ulm, Germany. 2 Institute for Human Genetics, RWTH Aachen University, Aachen, Germany. 3 Nephrology, University of Ulm, Ulm, Germany 4 Endocrinology 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 Ulm, Germany beate.karges@uniklinik-ulm.de 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

2 Heterozygous mutations of the POU A/homeodomain transcription factors hepatocyte nuclear factor (HNF)- 1 and HNF-1 cause maturity onset diabetes of the young (MODY) in humans (1). HNF-1 and HNF-1 act in a complex network of transcription factors regulating tissue-specific gene expression in the pancreas and other epithelial organs. Patients with mutations of HNF-1β (MODY5) are characterized by urogenital malformations, while extrapancreatic manifestations in patients with diabetes and HNF-1 mutations (MODY3) are not well known (2). History and examination After an occasional blood glucose reading of 13.4 mmo/l, the diagnosis of diabetes was established in a 13-year old girl. She was asymptomatic except for mild chronic lower abdominal discomfort. Her fasting glucose was 9.3 mmol/l, HbA1c was 8.5% (normal, %), and urine ketones were negative. After brief insulin treatment, she received glimepiride 0.5 mg/day, with a current HbA1c of 5.6%. Her father (Fig. 1), diagnosed with diabetes at the age of 27 years, has received insulin since the age of 38 years. The paternal grandmother had diabetes treated with glibenclamide until she died 20 years ago, aged 62 years. Two asymptomatic sisters of the index patient were diagnosed with diabetes at the age of 14 years and 19 years. Their fasting serum glucose was 7.3 and 6.8 mmol/l, and HbA1c was 6.0 and 6.1%, respectively. 2 hours after a 75 g oral glucose challenge, their glucose levels were 10.1 and 13.1 mmol/l. Fasting C-peptide was pmol/l in the index patient, and and pmol/l in her sisters; all had normal insulin sensitivity (HOMA 2.0, 1.7 and 1.8, respectively) and normal BMI (<25 kg/m 2 ). Liver, kidney and thyroid function tests including serum thyrotropin, free T 4 and T 3, creatinine, and other standard laboratory parameters were normal in all sisters, and serum islet cell autoantibodies and GAD65 antibodies were negative. Investigation Beta cell function was assessed after intravenous challenge with glucose (0.5 g/kg bolus) and L-arginine (0.7g/min for 30 min). In the index patient and her sisters, insulin secretion was significantly impaired after IV glucose (maximum serum insulin 223.8, and pmol/l, respectively). In contrast, there was a sustained insulin release after arginine infusion (maximum serum insulin 543.1, 363.0, and pmol/l, respectively). Abdominal ultrasound in the index patient showed agenesis of the right kidney and a didelphic uterus with hypoplastic right uterine horn and hemiatresia of the cervix, which was confirmed by hysteroscopy. In both of her sisters, no abnormalities of the urogenital system were identified by ultrasound. In the index patient and the younger sister, polycystic changes in both thyroid lobes with >50 cysts up to 4 mm in size were detected by high-resolution (12 MHz) ultrasound, and single thyroid cysts were identified in the father and the older sister. No cystic or other lesions in the liver were identified in any individual. HNF-1β sequence analysis in the index patient revealed a heterozygous genomic missense variant (c.1006c>g) in exon 4, resulting in the substitution of a highly conserved residue (p.his336asp) in the protein s transactivation domain. This novel variant was detected in 1 of 400 chromosomes in healthy individuals by denaturing high-performance liquid chromatography. Sequencing identified HNF-1β c.1006c>g in the younger sister and the father, but not in the older diabetic sister. Thus, this mutation could not exclusively account for the diabetes phenotype in this family. We next analyzed HNF-1 and identified a heterozygous genomic mutation (c.526+1delgtaa) in the canonic splice site of intron 2 in all diabetic individuals, but not in unaffected family members (Fig. 1). This novel mutation is predicted to result in aberrant HNF-1 splicing (3), with the introduction of a premature termination codon at amino acid position 194 and

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

4 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: , 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: , Wang M, Marin A: Characterization and prediction of alternative splice sites. Gene 366: , 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: , 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: , 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, 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: , 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: , de Felice M, di Lauro R: Thyroid development and its disorders: genetics and molecular mechanisms. Endocrine Reviews 25: ,

5 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. 5