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Hepatocyte Nuclear Factor-1 Gene Inactivation: Cosegregation between Liver Adenomatosis and Diabetes Phenotypes in Two Maturity-Onset Diabetes of the Young (MODY)3 Families

Department of Endocrinology (Y.R.), Department of Hepatology (T.D, A.F.), Department of Hepatic Surgery and Transplantation (L.C.), and Laboratory of Pathology (P.R.), Centre Hospitalier Universitaire (CHU) of Caen, 14033 Caen; Department of Pediatrics (R.C.) and Department of Gastroenterology (F.O.), CHU of Angers, 49033 Angers; Institut National de la Santé et de la Recherche Médicale U434 (E.J., J.Z.-R), CEPH-Fondation Jean Dausset, 75010 Paris; Laboratory of Anatomy, Cytology, and Pathology, Bicêtre Hospital, Le Kremlin Bicêtre 94275 (M.F.); and Laboratories of Molecular Biology (S.C., C.B.-C.) and Embryology and Cytogenetics (C.B.-C.), Saint-Antoine Hospital, 75012 Paris, France

Address all correspondence and requests for reprints to: Yves Reznik, M.D., Département d’Endocrinologie, Centre Hospitalier Universitaire Côte de Nacre, 14033 Caen, France. E-mail: reznik-y{at}chu-caen.fr.

	Abstract

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Heterozygous germline mutations of the hepatocyte nuclear factor (HNF)-1 are associated with maturity-onset diabetes of the young (MODY)3. Recently, the biallelic inactivation of the HNF-1 gene was reported in liver adenomas. We show the occurrence of liver adenomatosis in six MODY3-affected patients from two unrelated and large families. Liver adenomatosis was characterized by the presence of numerous adenomas within a normal hepatic parenchyma. The HNF-1 hot-spot germline mutation P291fs was identified in the two probands and in 16 relatives from the two families. The six patients affected by liver adenomatosis and diabetes exhibited the mutation. The analysis of liver-cell tumors from two affected patients evidenced the biallelic inactivation of HNF-1. The familial screening confirmed the clinical heterogeneity of the liver phenotype, from silent liver adenomatosis to fatal hemorrhage. These observations warrant the systematic screening for liver adenomatosis in MODY3 families to prevent its potentially deadly complications. Moreover, such screening may help to determine if a particular mutational spectrum of HNF-1 is associated with liver adenomatosis and to establish its prevalence in this frequent form of diabetes in the young adult.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
MATURITY-ONSET DIABETES of the young (MODY)3 is an autosomal dominant inherited form of nonketotic diabetes mellitus characterized by an early onset of diabetes before the age of 25 yr, a primary ß-cell dysfunction of variable intensity, and an altered renal tubular glucose reabsorption (1, 2, 3). Heterozygous mutations in the hepatocyte nuclear factor (HNF)-1 gene (also known as TCF1 gene) are associated with MODY3 and may account for 2–5% of non-insulin-dependent diabetes mellitus (4, 5).

Hepatocellular adenoma is a benign neoplasm usually developed as a solitary nodule but may occur as multiple or disseminated nodules in the so-called liver adenomatosis (6, 7). Liver adenomatosis is a rare disease, with only 38 cases reported until 2000 in the literature (7), but may be occult and thus underestimated (7, 8). Intratumoral or ip bleeding and possible malignant transformation have been reported as complications of liver adenomatosis (6, 7, 9, 10, 11). Liver adenomatosis was previously reported as a sporadic disease, whereas familial forms were scarce in the literature and were surprisingly associated with familial diabetes (7, 12). The involvement of a biallelic inactivation of HNF-1 in hepatic adenomas was recently reported, either by double somatic events or in association with a germline HNF-1 mutation. These results have stressed that HNF-1 associated with MODY3 met the genetic criteria of a classical tumor-suppressor gene (13).

We herein report the cosegregation of liver adenomatosis and diabetes phenotypes in two unrelated large French MODY3 families with the P291fs germline HNF-1 mutation. The somatic inactivation of the second HNF-1 allele in liver-cell adenomas was demonstrated from two affected patients. We point out the high variability of liver adenomatosis expression. The life-threatening potential of silent liver adenomatosis raises the question of its clinical screening in MODY3 patients.

	Patients and Methods

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Clinical investigation

The probands from families 1 and 2 presented with early-onset non-insulin-dependent diabetes associated with hepatomegaly. Liver nodules were discovered by noninvasive radiological imaging, and liver adenomatosis was confirmed by pathological diagnosis. These findings led to familial screening for diabetes and liver adenomatosis in both families. Criteria for the diagnosis of overt diabetes were based on repeated fasting plasma glucose over 126 mg/dl (7.0 mmol/liter) (14). Fasting hyperglycemia was defined as a fasting plasma glucose between 110 mg/dl (6.1 mmol/liter) and 125 mg/dl (6.9 mmol/liter). Screening for liver adenomatosis was based on liver ultrasonography and/or magnetic resonance imaging (MRI), with a diagnosis criterion of 10 or more liver nodules (6, 8). Pathological diagnosis was performed from surgical specimens.

Blood and tissue sample preparation

Peripheral blood samples were obtained from patients from families 1 and 2 who accepted the genetic analysis for MODY genes and signed informed written consent. Genomic DNA extraction was performed from peripheral blood leukocytes using a standardized phenol-chloroform procedure. Liver tumor tissue samples collected from two patients with proven liver adenomatosis by surgical biopsy were frozen immediately in liquid nitrogen and stored at -80 C. Tumor DNA was extracted using a salting-out procedure (15).

Molecular genetic studies

Germline HNF-1 mutation analysis. The 10 exons, exon-intron boundaries, and the promoter region of HNF-1 were screened for mutations in the two probands by direct sequencing of PCR products as previously described (4). The direct diagnosis of the mutation identified in the proband was then offered to relatives independently of their clinical status.

Somatic HNF-1 mutation analysis. The diagnosis of the identified germline mutation was performed by sequencing the corresponding PCR products in tumor DNA. The sequences of the 10 exons of HNF-1 with the analysis of 10 known coding single-nucleotide polymorphisms were subsequently performed for the search of a mutation in the second allele or a loss of heterozygosity as previously described (13).

	Results

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MODY phenotype

The familial history of diabetes and the age at diagnosis of diabetes in both families suggested that the two probands may have a MODY diabetes (Fig. 1; Table 1). The screening of HNF-1 associated with MODY3 was therefore initiated in these two subjects. The same P291fs (872_873insC) mutation located in exon 4 resulting from the insertion of a cytosine in the poly-C tract repeat and leading to a stop codon at position 316 was identified in the two probands. This mutation was subsequently searched in 26 relatives from both families and found in nine relatives from family 1 and seven relatives from family 2 (Fig. 1). Fourteen patients, including the two probands and 12 relatives presenting with an abnormal metabolic phenotype, inherited the germline P291fs mutation. Metabolic disturbances associated with the HNF-1 mutation were variable, including fasting hyperglycemia, gestational diabetes, and overt diabetes (Fig. 1). Four patients from both families were carriers of the HNF-1 mutation but still exhibited normal fasting glycemia (Fig. 1). These features are consistent with the variable expressivity of glycemic abnormalities and incomplete penetrance observed within and between MODY3 families (16).

View larger version (22K): [in this window] [in a new window]   FIG. 1. Pedigrees of families 1 and 2. Patients are identified by generation numbers (roman numerals on the left) and individual numbers within each generation below the symbol. Probands are indicated by arrows. The second line under the symbols corresponds to the current age of patients. Current age in parentheses corresponds to the age of death. The HNF-1 genotype of each patient tested is indicated on the third line (M+, patients carrying the P291fs mutation; M–, patients not carrying the mutation).

View this table: [in this window] [in a new window]   TABLE 1. Clinical characteristics of patients presenting with liver adenomatosis and diabetes mellitus and carrying the P291fs HNF-1 mutation

Liver adenomatosis was diagnosed in the two probands presenting with early-onset non-insulin-dependent diabetes and hepatomegaly. All relatives screened for mutations in HNF-1 were subsequently investigated, and liver adenomatosis was discovered in four of them. All the patients with liver adenomatosis had diabetes. Their clinical characteristics were highly variable (Table 1). In one patient (IV-10, family 1), liver adenomatosis was revealed at age 16 by a dramatic ip bleeding that was fatal. In the three others, liver adenomatosis was occult with no liver enzyme disturbance and was therefore diagnosed by ultrasonography screening. Long-term follow-up was available in four patients with liver adenomatosis (Table 1). In the proband of family 1, progressive growth of hepatic nodules and intrahepatic adenoma hemorrhage after a 15-yr period led to liver transplantation. In two patients, no progression of the disease was observed after partial hepatectomy. The last one did not exhibit any evolution after a 7-yr period of follow-up. Among the other relatives, two patients partially explored in family 1 presented with phenotypic features consistent with liver adenomatosis; patient II-1 carried the germline mutation, had diabetes, and exhibited a solitary hepatic nodule on ultrasonography imaging. His mother (I-2) had died of an intraabdominal hemorrhage at age 23. Patients with no HNF-1 mutation were also screened, and none exhibited liver ultrasonographic abnormalities.

The diagnosis of liver adenomatosis was based on pathological specimens in five patients and on liver imaging in one (patient III-8, family 2). In the latter, multiple (>10) hyperechoic nodules were seen at liver ultrasonography, the four largest appearing on MRI imaging as isointense T1 and hypointense T2-weighted images with a transitory enhancement of hepatic arterial phase after bolus administration of contrast material (8). Pathological examination was performed from either hepatectomy specimens in four cases or liver biopsy in one case. Diffuse liver adenomatosis was observed in all liver specimens. The striking feature was the macroscopic presence of innumerable tan colored clusters varying in size from less than a millimeter to several centimeters (Fig. 2A). Foci of hemorrhage and necrosis were seen in the larger adenomas. Microscopically, a patchwork of adenomatous liver cell clusters with irregular jagged edges were detected in grossly normal appearance of the liver. In all adenomas, the hepatocytes were both swollen and vacuolated with an excessive cytoplasmic and nuclear lipid and glycogen accumulation (Fig. 2B). Neither estrogen nor progesterone receptor expression was noted in liver nuclei.

View larger version (62K): [in this window] [in a new window]   FIG. 2. A, Section of the liver at necropsy in a 16-yr-old woman (patient IV-10, family 1) with a massive liver adenomatosis at diagnosis. Innumerable nodules, tan colored and varying in size from 0.5–15 cm (arrows), were detected. Nodules were sharply circumscribed from the adjacent liver but not encapsulated. B, An adenomatous liver cell cluster. The cells were large and vacuolated. Normal liver cells are shown on the background with a portal tract (hematoxylin and eosin; magnification, x20). Inset, Fatty liver cells with glycogenated nuclei confirmed on oil red O and periodic acid Schiff stains (arrow) (hematoxylin, eosin, and safran, magnification x100).

Biallelic inactivation of HNF-1

Two patients, IV-11 and III-10 from families 1 and 2, respectively (Fig. 1), were investigated for somatic alterations of HNF-1 in their liver-cell tumors. A biallelic inactivation of HNF-1 was demonstrated in both. In the first, two mutations in tumor DNA were identified, the P291fs (872_873insC) mutation in one allele and an in-frame deletion of three amino acids F277_H279del (829_837del) located in the homeodomain of HNF-1 in the second allele. In the second, the germline P291fs mutation was observed in one allele and the HNF-1 gene deletion in the second allele.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
We herein describe the occurrence of liver adenomatosis in six MODY3 patients from two large families, in whom the HNF-1 gene alterations were identified as a heterozygous germline mutation in peripheral blood cells and detected as a biallelic HNF-1 gene inactivation in the liver-cell adenomas from two affected patients. In the present report, 14 patients were carriers of a mutation in HNF-1, and six of them were affected by liver adenomatosis. Clinical expression of liver adenomatosis at diagnosis was highly variable, from fatal hemorrhage in a 16-yr-old girl to silent expression in other patients, thus illustrating the wide range of age at diagnosis, from adolescence to late onset after age 50. The course of the disease was either stable or associated with complications that required surgery. Liver adenomatosis was absent in eight diabetic relatives identified with a germline HNF-1 mutation, suggesting an incomplete penetrance of the phenotypic trait. Of note, there was no evidence that the liver phenotype worsened the severity of diabetes. No additional environmental factor was evidenced in patients with the liver phenotype, in contrast with solitary liver adenoma, where the use of contraceptives was found in approximately 90% of the female affected patients in Western countries (6). Our data do not support such a role of oral contraceptives in patients with HNF-1 alterations.

This report confirms the biallelic inactivation of HNF-1, consisting of either two distinct mutations or a mutation associated with the HNF-1 gene deletion, in patients with liver adenomatosis and MODY3 diabetes. In our two families, the heterozygous germline mutation was the P291fs mutation, the most common mutation identified in patients with MODY3 diabetes (17, 18). Interestingly, the HNF-1 mutations observed by Bluteau et al. in liver adenomas were either frameshift or nonsense mutations spread over the entire coding sequence of HNF-1 or missense mutations located in the homeodomain (13). In contrast, germline HNF-1 mutations found in patients with MODY3 diabetes are mainly missense mutations spread throughout the entire sequence of HNF-1 (19). These findings raise the question of a particular mutational spectrum that may be preferentially involved in HNF-1 inactivation associated with the development of liver adenomatosis.

HNF-1 is a transcription factor expressed in various tissues, including liver, pancreas, kidney, and the digestive tract. Its role in liver function and liver proliferation is not yet elucidated. Animal studies have shown that HNF-1 complete inactivation in mice resulted in a marked liver enlargement and hepatic dysfunction (20, 21). Experimental liver carcinogenesis in the rat is associated with a decrease in HNF-1 expression in preneoplastic nodules, which suggests its role in cellular growth control of liver nodules (22).

In summary, the identification of two multiplex MODY3 families enabled us to confirm the cosegregation of liver adenomatosis with the biallelic inactivation of HNF-1. The study of affected relatives underlined the wide variability of liver adenomatosis expression and the possible latency of the disease for several decades. Due to potentially severe complications, these observations warrant the systematic screening of liver adenomatosis by noninvasive liver imaging in MODY3 families and careful follow-up of the disease by multidisciplinary teams including hepatologists, radiologists, diabetologists, and geneticians. Additional studies may help to establish the prevalence of liver adenomatosis in MODY3, the most frequent monogenic form of diabetes in young adults, and to evidence environmental factors or the involvement of modifier genes that may account for the variable clinical expression of liver adenomatosis.

	Acknowledgments

 
We are indebted to J. P. Saint André, M.D. (Laboratoire d’anatomo-pathologie, CHU d’Angers), and P. Bioulac-Sage, M.D. (Laboratoire d’anatomo-pathologie, CHU Pellegrin, Bordeaux), for their contribution to the pathological diagnosis and X. Rialland, M.D. (Département de pédiatrie, CHU d’Angers), F. Gauthier and S. Branchereau (Service de chirurgie digestive, CHU du Kremlin-Bicêtre, Paris), and E. Jacquemin (Service d’hépatologie infantile, CHU du Kremlin-Bicêtre, Paris) for their contribution to the clinical management. We thank the families for their participation in the study.

	Footnotes

 
Abbreviations: MODY, Maturity-onset diabetes of the young; MRI, magnetic resonance imaging.

Received September 5, 2003.

Accepted November 26, 2003.

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