- Email: [email protected]
Genetic variants of hepatocyte nuclear factor-1β in Chinese young-onset diabetic patients with nephropathy
Journal of Diabetes and Its Complications 17 (2003) 369 – 373
Genetic variants of hepatocyte nuclear factor-1b in Chinese young-onset diabetic patients with nephropathy W.Y. Soa,*, Maggie C.Y. Nga, Yukio Horikawab, Pal R. Njølstadb, June K.Y. Lia, Ronald C.W. Maa, Graeme I. Bellb, Juliana C.N. Chana a
Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR, China b Howard Hughes Medical Institute, University of Chicago, Chicago, IL 60637, USA Received 16 April 2002; received in revised form 29 June 2002; accepted 16 August 2002
Abstract Background: In Hong Kong, the prevalence of diabetes is estimated to be 2% in the young population. In the diabetic population, 30% of patients have diagnosis before the age of 40 years. Besides, 30% of young diabetic patients have varying degrees of albuminuria [Janus et al., 2000; Cockram et al., 1993; Chan et al., 1995]. Mutations in the gene encoding the hepatocyte nuclear factor (HNF)-1b are associated with a subtype of maturity-onset diabetes of the young (MODY 5) characterized by urogenital abnormalities. We examined 74 unrelated Chinese subjects with young-onset diabetes complicated by nephropathy for variants in this gene. Methods: The HNF-1b gene was screened by direct sequencing and the functional properties of wild-type and mutant proteins were analyzed by transactivation analysis. Results: A novel variant in exon 3 (E260D) was found in one patient. Extended family analysis revealed four other siblings carrying this variant. One subject had diabetes and another had impaired glucose tolerance. Another sibling had microalbuminuria but normal glucose tolerance. Transfection studies showed insignificant differences in transactivation ability between wild-type and mutated HNF-1b. A silent polymorphism Q378Q was identified in another unrelated subject. Conclusions: These results suggest genetic variants in HNF-1b are not a common cause of young-onset diabetes or diabetic nephropathy in Chinese, but may modify disease manifestation and progression. Other potential candidate genes should be looked for to account for the high prevalence of young-onset diabetes and nephropathy in this population. D 2003 Elsevier Inc. All rights reserved. Keywords: Genetics; Diabetes; HNF-1b; Maturity-onset diabetes of young; Nephropathy; Transcription; Chinese
1. Introduction Recent studies have identified at least six genes responsible for maturity-onset diabetes of the young (MODY) characterized by autosomal dominant inheritance, early onset (usually before 25 years of age), and a primary defect in pancreatic b-cell function. Apart from MODY 2, due to mutations in glucokinase gene, the other forms of MODY are due to heterozygous mutations in transcription factors mainly belonging to the hepatocyte nuclear factor (HNF) family (Fajans, Bell, & Polonsky, 2001). These transcription factors form a network with HNF-1b functioning as homo- or heterodimers with HNF-1a (Rey-Campos, Chouard, Yaniv, & Cereghini, 1991) and they are expressed
* Corresponding author. Tel.: +852-2632-3138; fax: +852-2632-3108. E-mail address: [email protected] (W.Y. So). 1056-8727/03/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1056-8727(02)00221-0
sequentially in renal tubules with possible roles in various critical stages of development of renal tubules (Cereghini, Ott, Power, & Maury, 1992). Against this background, there is marked heterogeneity in the phenotypes of patients with mutations in different MODY genes in terms of metabolic and renal manifestations. Mutations in HNF-1b are associated with MODY5 (Horikawa et al., 1997), characterized by mild diabetes but increased susceptibility to severe renal disease and other urogenital malformations. Knock-out mice model shows that deficiency of HNF-1b is associated with pancreatic b-cell and renal dysfunction (Bingham et al., 2000; Horikawa et al., 1997; Lindner et al., 1999; Tomura et al., 1999; Weng et al., 2000). On the other hand, patients with mutations in HNF-1a causing MODY3 have subtle renal disease with a low renal threshold for glucose (Menzel et al., 1998). Two epidemiological studies have shown that the prevalence of diabetes is reaching epidemic proportion among
370
W.Y. So et al. / Journal of Diabetes and Its Complications 17 (2003) 369–373
Hong Kong Chinese and continues to rise. In subjects under the age of 40 years, the prevalence of diabetes has been reported to be 1% in 1990 (Cockram et al., 1993), rising to 2% in 1995 (Janus et al., 2000). In both community and clinic-based populations, 20– 30% of our diabetic patients have diagnosis before the age of 40 years (Chan, Cheung, Swaminathan, Nicholls, & Cockram, 1993). However, classical autoimmune type 1 diabetes accounts for only approximately 10% in young Chinese diabetic population. There is increasing evidence that genetic factors interact with environmental and lifestyle factors, mainly reflected by obesity, to give rise to early-onset diabetes. As many as 50% of young Chinese diabetic patients have family history of diabetes. In a pilot study, the prevalence of mutations in glucokinase and HNF-1a genes in our young Chinese type 2 diabetic patients is 3% and 5%, respectively, while mutation in HNF-4a is uncommon (Chan, 2000; Ng et al., 1999). Hence, the cause of diabetes in the majority of young patients remains to be identified. Besides, 30– 50% of our young diabetic patients have significant albuminuria despite short disease duration of less than 5 years (Ko, Chan, Lau, & Cockram, 1999). End stage renal disease is the cause of death in 10 – 30% of Chinese type 2 diabetic patients (Colhoun et al., 2001). This is in contrast to Caucasians in whom ischaemic heart disease is the leading cause of death. Since genetic factors play an important role in the development of diabetic nephropathy (Rippin, Patel, & Bain, 2001), we hypothesized that HNF-1b mutation may be implicated in the development of young-onset diabetes with nephropathy. To test this hypothesis, we screened HNF-1b for mutations in a group of 74 Chinese unrelated subjects with familial early-onset type 2 diabetes who had albuminuria in their early course of disease.
2. Patients and methods A total of 74 unrelated Chinese subjects were studied. They presented with type 2 diabetes (1995 WHO criteria) before the age of 40 years and had at least one first degree relative affected with diabetes. A random spot urine sample followed by a timed collection (4- or 24-h) was used to document albuminuria. All of them had diabetic nephropathy (based on two out of three urine collections) defined as 24-h urinary albumin excretion (UAE) 300 mg/day, 4-h urinary albumin excretion rate (AER) 200mg/min, and/or spot urinary albumin – creatinine ratio (ACR) 25 mg/ mmol (Mogensen et al., 1995). Thirty-one percent of them met the minimal criteria for MODY (age of diagnosis before 25 years of age and presence of diabetes in two consecutive generations). These patients were selected from a Diabetes Registry containing detailed clinical and biochemical information of 2000 cases recruited between February 1995 and August 1997 from the Diabetes and Endocrine Center of the Prince of Wales Hospital, the teaching hospital of the Chinese University of Hong Kong.
The mean age of onset for the subjects (47% male) was 30 years (range 10 –40 years) and the mean age at the time of assessment was 40 years (range 22– 60 years). Fifty-three percent of patients were treated with insulin with or without oral drugs and 24.3% were on oral drugs only. The remaining 23% patients were on diet. The mean ( ± S.D.) HbAlc was 8.6 ± 2.1%. Their geometric mean plasma creatinine was 118.0mmol/l (range 35 – 1293mmol/l) and AER was 601.8 ± 3.46mg/min. Thirteen percent were treated with inhibitors of the renin – angiotensin – aldosterone system. Thirty-three percent of patients had clinical evidence of diabetic retinopathy and 6.4% had macrovascular complications, namely ischaemic heart disease, cerebrovascular accident, or significant peripheral vascular disease. The study was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong, and informed consent for DNA donation was obtained from each subject. 2.1. Screening for HNF-1 mutations The minimal promoter region, flanking introns and exons of the HNF-1b gene were screened for mutations by direct sequencing of polymerase chain reaction (PCR) products, as described previously (Horikawa et al., 1997). 2.2. Transfection studies The missence mutation was introduced into a DNA fragment, which was cloned into a plasmid, pcDNA3.1HNF-1b, encoding the type A form of human HNF-1b, as described previously (Lindner et al., 1999). The reporter gene construct, pGL3-RA, was prepared using methodology, as reported previously (Bjorkhaug et al., 2000). HeLa cells were grown and transfected with DNA either of the wild-type or mutant type of HNF-1b-containing plasmid, the reporter gene pGL3-RA, pRL-SV40 as control as well as pcDNA 3.1+ (Bjorkhaug et al., 2000; Lindner et al., 1999). The transactivation activity of the normal and mutant HNF1b proteins was measured (Bjorkhaug et al., 2000). 2.3. Clinical studies All patients underwent a structured assessment including documentation of family history, as well as clinical and biochemical characteristics. All patients had a random spot urine sample followed by a timed urinary collection, either 4- or 24-h, for measurement of UAE. For patients confirmed to have HNF-1b mutation, ultrasonographic examination of kidney was carried out to screen for renal cysts and other urogenital abnormalities, which have been reported to be associated with the mutations. Family members of probands identified with HNF-1b mutation, if available, were recruited. All of them underwent 75-g oral glucose tolerance test (OGTT) and ultrasonography. Direct sequencing to screen for mutations was performed in all family members who agreed to undergo assessment.
W.Y. So et al. / Journal of Diabetes and Its Complications 17 (2003) 369–373
371
Fig. 1. Pedigree of Hong Kong Chinese family HK34. The proband was labelled with arrow and other subjects studied are labelled (a) – (f ). Details are described in text. Subjects with diabetes are indicated by filled symbols. Genotype: N, normal allele; M, E260D allele. BMI: body mass index (kg/m2), ACR: albumin – creatinine ratio (mg/mmol), plasma creatinine in mmol/l.
Plasma glucose concentration was measured by a glucose oxidase method (Diagnostic Chemicals, Charlottetown, Prince Edward Island, Canada). HbAlc was measured by gel electrophoresis (Ciba Corning Diagnostic, Palo Alto, USA). Data are expressed as mean ± S.D. or geometric mean ± antilog S.D. as appropriate.
3. Results Of the 74 patients, a missense genetic variant in exon 3 (E260D, GAG ! GAC) was identified in a male patient (HK34) who was diagnosed to have diabetes at the age of 36 years. At the time of examination in 1995, his age was 49
years, with a body mass index (BMI) of 30.2 kg/m2 and blood pressure of 162/100 mm Hg. His 24-h UAE was 832.4 mg/day and plasma creatinine was 101mmol/l (normal range 44 –107mmol/l). Clinically, there was no evidence of other glomerulopathy. His HbAlc was 6.9% (normal range 5.1 – 6.4%) and there was no other diabetic complications including retinopathy. He was treated with oral agents. The same mutation was also present in 2 of 150 (1.3%) unrelated nondiabetic Chinese subjects who had normal renal function. Screening of the other family members of HK34 revealed the same variant in four of the siblings available, in whom three of them (aged 38 (subject c), 34 (subject e), and 32 (subject f) years, respectively) had normal glucose tolerance using the 1998 WHO criteria (Fig. 1). Subject c and f had
Fig. 2. Result of transfection study using transcriptional activation by epitope-tagged wild-type and E260D of HNF-1b. Details of method are described in text. There was no significant difference in activity between the wild-type and the E260D variant. The addition of the wild-type and E260D variant resulted in additional effect in a dose-dependent manner.
372
W.Y. So et al. / Journal of Diabetes and Its Complications 17 (2003) 369–373
normoalbuminuria and their BMI were 19.7 and 28.4 kg/m2, respectively. Subject e had microalbuminuria with a spot urine ACR of 5.0 mg/mmol (normal range < 3.5 mg/mmol), plasma creatinine of 63 mmol/l, and BMI of 20.8 kg/m2. One sibling (subject d) was found to have diabetes based on 75-g OGTT at the age of 36 years (plasma glucose at 0 and 120 min were 4.9 and 13.2 mmol/l, respectively). She had a BMI of 23.5 kg/m2 and had normoalbuminuria with plasma creatinine of 57mmol/l. The other sibling (subject b) had impaired glucose tolerance (IGT) (plasma glucose of 5.8 and 9.8 mmol/l at 0 and 120 min, respectively) at age of 40 years. He had a BMI of 26.2 kg/m2 and a plasma creatinine of 68 mmol/l. The proband’s mother had unknown status of glucose tolerance and she died at the age of 65 years due to heart disease. Ultrasonographic studies revealed multiple renal cysts and fatty liver in the proband. Renal cysts were also identified in subject f and there was no urogenital abnormalities identified in subjects c, d, and e (Fig. 1). Transfection studies revealed that there was insignificant difference for stimulated transcription of a firefly luciferase reporter gene linked to the HNF-1 binding site of the rat albumin gene (Fig. 2). Also, there was no inhibition of HNF-1b activity when cells were transfected with equivalent amounts of wild-type and mutant HNF-1b expression vectors, implying the mutation did not function in a dominant-negative manner. In addition, a polymorphism in exon 5 (Q378Q, CAG ! CAA) was identified in a 50-year-old patient who had diabetes since 37 years of age. He was treated with an oral agent. HbAlc was 9.4% and plasma creatinine was 105 mmol/l. The polymorphism Q378Q was not present in 105 unrelated nondiabetic Chinese subjects. There were no other mutations nor polymorphisms identified in the cohort.
4. Discussion According to the WHO Multinational Study of Vascular Disease in Diabetes findings, Chinese diabetic subjects have increased risk of developing renal disease. In contrast to Caucasian populations where cardiovascular disease is the leading cause of death, 30– 50% of Chinese diabetic patients die from end stage renal disease (Colhoun et al., 2001). Furthermore, 30– 50% of Chinese patients have albuminuria, which predicts the decline of renal function (Chan et al., 1995). Mutations in HNF-1b are associated with MODY5, which is associated with varying severity of glycaemia and renal failure. Apart from multiple renal cysts, which are usually present before the onset of diabetes (Horikawa et al., 1997; Lindner et al., 1999; Tomura et al., 1999), severe urogenital developmental abnormalities were identified in a Norwegian pedigree, suggesting a broad spectrum of clinical syndrome beyond glucose intolerance associated with this mutation (Lindner et al., 1999). Based on knockout mice experiment, HNF-1b, which is expressed
in renal tubules, appears to play an important role in its development (Barbacci et al., 1999; Bingham et al., 2001; Coffinier, Thepot, Babinet, Yaniv, & Barra, 1999). In light of the high prevalence of young-onset diabetes and relatively high prevalence of nephropathy (30%), we examined the potential impact of HNF-1b gene mutations or polymorphisms in our young diabetic patients with clinical evidence of renal disease. We hypothesize that the gene may be a factor in the development of diabetes and may increase the susceptibility of developing nephropathy. The novel missense variant HNF-1b E260D identified in our patient affects a highly conserved amino acid (human to Xenopus) in the DNA binding domain. Changing the amino acid from glutamic to aspartic acid results, however, in only a minor change in the carbon chain involved in protein – protein and protein – DNA interaction and with minimal changes in the protein charge. Besides, our transfection studies suggest only a minor effect of the variant on protein function. The proband carrying the mutation had macroalbuminuria with multiple renal cysts. Age of onset of diabetes was 36 years of age and he was treated with oral drugs. Two of the siblings (subjects d and b) were carriers of the mutation, with either diabetes (subject d) and impaired glucose tolerance (subject b). Both were obese according to the WHO Western Pacific Region criteria for Asian populations ( 25 kg/m2), but they had no renal or urogenital abnormalities in terms of albuminuria and on ultrasonographic imaging. The other siblings carrying the mutation had normal glucose tolerance although 2 of them had either albuminuria (subject e) or renal cysts on ultrasound imaging (subject f). Subject c, who had a BMI of 19.7 kg/m2, had normal glucose tolerance and normoalbuminuria. The heterogeneity of the phenotype in terms of glycaemia and urogenital manifestations together with results from the transfection studies suggested that E260D mutation might not play a major role in the development of diabetes or nephropathy. Nonetheless, it is plausible that it might interact with other modifying genes and/or risk factor such as raised blood pressure, increased plasma lipid or blood glucose to increase the risk of development of diabetes and its complications. To test this hypothesis, screening of the variant in a larger population will be required. Based on our pilot study, the frequency of HNF-1b variant in Chinese patients with familial early-onset type 2 diabetes was 1.4%. This finding is in agreement with the reported 2.5% prevalence in Japanese type 2 diabetic patients with early-onset disease (Horikawa et al., 1997). Given the low frequency of these genetic variants in both diabetic and nondiabetic subjects in different ethnic groups including Chinese, the heterogeneity of phenotypes in carriers and insignificant results from the transfection study, we concluded that HNF-1b genetic variants are not the major causes of early-onset type 2 diabetes or MODY but might have modifying effects. The clinical significance regarding its associations with renal manifestation in terms
W.Y. So et al. / Journal of Diabetes and Its Complications 17 (2003) 369–373
of either albuminuria or renal cysts also requires further elucidation (Beards et al., 1998; Weng et al., 2000).
References Barbacci, E., Reber, M., Ott, M. O., Breillat, C., Huetz, F., & Cereghini, S. (1999). Variant hepatocyte nuclear factor 1 is required for visceral endoderm specification. Development, 126, 4795 – 4805. Beards, F., Frayling, T., Bulman, M., Horikawa, Y., Allen, L., Appleton, M., Bell, G. I., Ellard, S., & Hattersley, A. T. (1998). Mutations in hepatocyte nuclear factor 1beta are not a common cause of maturity-onset diabetes of the young in the UK. Diabetes, 47, 1152 – 1154. Bingham, C., Bulman, M. P., Ellard, S., Allen, L. I., Lipkin, G. W., Hoff, W. G., Woolf, A. S., Rizzoni, G., Novelli, G., Nicholls, A. J., & Hattersley, A. T. (2001). Mutations in the hepatocyte nuclear factor1beta gene are associated with familial hypoplastic glomerulocystic kidney disease. American Journal of Human Genetics, 68, 219 – 224. Bingham, C., Ellard, S., Allen, L., Bulman, M., Shepherd, M., Frayling, T., Berry, P. J., Clark, P. M., Lindner, T., Bell, G. I., Ryffel, G. U., Nicholls, A. J., & Hattersley, A. T. (2000). Abnormal nephron development associated with a frameshift mutation in the transcription factor hepatocyte nuclear factor-1 beta. Kidney International, 57, 898 – 907. Bjorkhaug, L., Ye, H., Horikawa, Y., Sovik, O., Molven, A., & Njolstad, P. R. (2000). MODY associated with two novel hepatocyte nuclear factor-1alpha loss-of-function mutations (P112L and Q466X). Biochemical and Biophysical Research Communications, 279, 792 – 798. Cereghini, S., Ott, M. O., Power, S., & Maury, M. (1992). Expression patterns of vHNF1 and HNF1 homeoproteins in early postimplantation embryos suggest distinct and sequential developmental roles. Development, 116, 783 – 797. Chan, J. C. (2000). Heterogeneity of diabetes mellitus in the Hong Kong Chinese population. The Chinese University of Hong Kong-Prince of Wales Hospital Diabetes Research and Care Group. Hong Kong Medical Journal, 6, 77 – 84. Chan, J. C., Cheung, C. K., Cheung, M. Y., Swaminathan, R., Critchley, J. A., & Cockram, C. S. (1995). Abnormal albuminuria as a predictor of mortality and renal impairment in Chinese patients with NIDDM. Diabetes Care, 18, 1013 – 1016. Chan, J. C., Cheung, C. K., Swaminathan, R., Nicholls, M. G., & Cockram, C. S. (1993). Obesity, albuminuria and hypertension among Hong Kong Chinese with non-insulin-dependent diabetes mellitus (NIDDM). Postgraduate Medical Journal, 69, 204 – 210. Cockram, C. S., Woo, J., Lau, E., Chan, J. C., Chan, A. Y., Lau, J., Swaminathan, R., & Donnan, S. P. (1993). The prevalence of diabetes mellitus and impaired glucose tolerance among Hong Kong Chinese adults of working age. Diabetes Research and Clinical Practice, 21, 67 – 73. Coffinier, C., Thepot, D., Babinet, C., Yaniv, M., & Barra, J. (1999). Essential role for the homeoprotein vHNF1/HNF1beta in visceral endoderm differentiation. Development, 126, 4785 – 4794. Colhoun, H. M., Lee, E. T., Bennett, P. H., Lu, M., Keen, H., Wang, S. L.,
373
Stevens, L. K., & Fuller, J. H. (2001). Risk factors for renal failure: the WHO Mulinational Study of Vascular Disease in Diabetes. Diabetologia, 44 (Suppl. 2), S46 – S53. Fajans, S. S., Bell, G. I., & Polonsky, K. S. (2001). Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young. New England Journal of Medicine, 57, 971 – 980. Horikawa, Y., Iwasaki, N., Haram, N., Furuta, H., Hinokio, Y., Cockburn, B. N., Lindner, T., Yamagata, K., Ogara, M., Tomonaga, O., Kuroki, H., Kasahara, T., Iwamoto, Y., Bell, GI. (1997). Mutation in hepatocyte nuclear factor-1 beta gene (TCF2) associated with MODY. Nature Genetics, 17, 384 – 385. Janus, E. D., Watt, N. M., Lam, K. S., Cockram, C. S., Siu, S. T., Liu, L. J., & Lam, T. H. (2000). The prevalence of diabetes, association with cardiovascular risk factors and implications of diagnostic criteria (ADA 1997 and WHO 1998) in a 1996 community-based population study in Hong Kong Cardiovascular Risk Factor Steering Committee, American Diabetes Association. Diabetic Medicine, 17, 741 – 745. Ko, G. T., Chan, J. C., Lau, M., & Cockram, C. S. (1999). Diabetic microangiopathic complications in young Chinese diabetic patients: a clinicbased cross-sectional study. Journal of Diabetes and its Complications, 13, 300 – 306. Lindner, T. H., Njolstad, P. R., Horikawa, Y., Bostad, L., Bell, G. I., & Sovik, O. (1999). A novel syndrome of diabetes mellitus, renal dysfunction and genital malformation associated with a partial deletion of the pseudo-POU domain of hepatocyte nuclear factor-1beta. Human Molecular Genetics, 8, 2001 – 2008. Menzel, R., Kaisaki, P. J., Rjasanowski, I., Heinke, P., Kerner, W., & Menzel, S. (1998). A low renal threshold for glucose in diabetic patients with a mutation in the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene. Diabetic Medicine, 15, 816 – 820. Mogensen, C. E., Vestbo, E., Poulsen, P. L., Christiansen, C., Damsgaard, E. M., EiskjÆr, H., FrØland, A., Hansen, K. W., Nielsen, S., & Pedersen, M. M. (1995). Microalbuminuria and potential confounders. Diabetes Care, 18, 572 – 581. Ng, M. C., Cockburn, B. N., Lindner, T. H., Yeung, V. T., Chow, C. C., So, W. Y., Li, J. K., Lo, Y. M., Lee, Z. S., Cockram, C. S., Critchley, J. A., Bell, G. I., & Chan, J. C. (1999). Molecular genetics of diabetes mellitus in Chinese subjects: identification of mutations in glucokinase and hepatocyte nuclear factor-1alpha genes in patients with early-onset type 2 diabetes mellitus/MODY. Diabetic Medicine, 16, 956 – 963. Rey-Campos, J., Chouard, T., Yaniv, M., & Cereghini, S. (1991). vHNF1 is a homeoprotein that activates transcription and forms heterodimers with HNF1. EMBO Journal, 10, 1445 – 1457. Rippin, J. D., Patel, A., & Bain, S. C. (2001). Genetics of diabetic nephropathy. Best Practice Research and Clinical Endocrinology and Metabolism, 15, 345 – 358. Tomura, H., Nishigori, H., Sho, K., Yamagata, K., Inoue, I., & Takeda, J. (1999). Loss-of-function and dominant-negative mechanisms associated with hepatocyte nuclear factor-1beta mutations in familial type 2 diabetes mellitus. Journal of Biological Chemistry, 274, 12975 – 12978. Weng, J. P., Lehto, M., Forsblom, C., Huang, X., Li, H., & Groop, L. C. (2000). Hepatocyte nuclear factor-1 beta (MODY5) gene mutations in Scandinavian families with early-onset diabetes or kidney disease or both. Diabetologia, 43, 131 – 132.
Genetic variants of hepatocyte nuclear factor-1b in Chinese young-onset diabetic patients with nephropathy W.Y. Soa,*, Maggie C.Y. Nga, Yukio Horikawab, Pal R. Njølstadb, June K.Y. Lia, Ronald C.W. Maa, Graeme I. Bellb, Juliana C.N. Chana a
Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR, China b Howard Hughes Medical Institute, University of Chicago, Chicago, IL 60637, USA Received 16 April 2002; received in revised form 29 June 2002; accepted 16 August 2002
Abstract Background: In Hong Kong, the prevalence of diabetes is estimated to be 2% in the young population. In the diabetic population, 30% of patients have diagnosis before the age of 40 years. Besides, 30% of young diabetic patients have varying degrees of albuminuria [Janus et al., 2000; Cockram et al., 1993; Chan et al., 1995]. Mutations in the gene encoding the hepatocyte nuclear factor (HNF)-1b are associated with a subtype of maturity-onset diabetes of the young (MODY 5) characterized by urogenital abnormalities. We examined 74 unrelated Chinese subjects with young-onset diabetes complicated by nephropathy for variants in this gene. Methods: The HNF-1b gene was screened by direct sequencing and the functional properties of wild-type and mutant proteins were analyzed by transactivation analysis. Results: A novel variant in exon 3 (E260D) was found in one patient. Extended family analysis revealed four other siblings carrying this variant. One subject had diabetes and another had impaired glucose tolerance. Another sibling had microalbuminuria but normal glucose tolerance. Transfection studies showed insignificant differences in transactivation ability between wild-type and mutated HNF-1b. A silent polymorphism Q378Q was identified in another unrelated subject. Conclusions: These results suggest genetic variants in HNF-1b are not a common cause of young-onset diabetes or diabetic nephropathy in Chinese, but may modify disease manifestation and progression. Other potential candidate genes should be looked for to account for the high prevalence of young-onset diabetes and nephropathy in this population. D 2003 Elsevier Inc. All rights reserved. Keywords: Genetics; Diabetes; HNF-1b; Maturity-onset diabetes of young; Nephropathy; Transcription; Chinese
1. Introduction Recent studies have identified at least six genes responsible for maturity-onset diabetes of the young (MODY) characterized by autosomal dominant inheritance, early onset (usually before 25 years of age), and a primary defect in pancreatic b-cell function. Apart from MODY 2, due to mutations in glucokinase gene, the other forms of MODY are due to heterozygous mutations in transcription factors mainly belonging to the hepatocyte nuclear factor (HNF) family (Fajans, Bell, & Polonsky, 2001). These transcription factors form a network with HNF-1b functioning as homo- or heterodimers with HNF-1a (Rey-Campos, Chouard, Yaniv, & Cereghini, 1991) and they are expressed
* Corresponding author. Tel.: +852-2632-3138; fax: +852-2632-3108. E-mail address: [email protected] (W.Y. So). 1056-8727/03/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1056-8727(02)00221-0
sequentially in renal tubules with possible roles in various critical stages of development of renal tubules (Cereghini, Ott, Power, & Maury, 1992). Against this background, there is marked heterogeneity in the phenotypes of patients with mutations in different MODY genes in terms of metabolic and renal manifestations. Mutations in HNF-1b are associated with MODY5 (Horikawa et al., 1997), characterized by mild diabetes but increased susceptibility to severe renal disease and other urogenital malformations. Knock-out mice model shows that deficiency of HNF-1b is associated with pancreatic b-cell and renal dysfunction (Bingham et al., 2000; Horikawa et al., 1997; Lindner et al., 1999; Tomura et al., 1999; Weng et al., 2000). On the other hand, patients with mutations in HNF-1a causing MODY3 have subtle renal disease with a low renal threshold for glucose (Menzel et al., 1998). Two epidemiological studies have shown that the prevalence of diabetes is reaching epidemic proportion among
370
W.Y. So et al. / Journal of Diabetes and Its Complications 17 (2003) 369–373
Hong Kong Chinese and continues to rise. In subjects under the age of 40 years, the prevalence of diabetes has been reported to be 1% in 1990 (Cockram et al., 1993), rising to 2% in 1995 (Janus et al., 2000). In both community and clinic-based populations, 20– 30% of our diabetic patients have diagnosis before the age of 40 years (Chan, Cheung, Swaminathan, Nicholls, & Cockram, 1993). However, classical autoimmune type 1 diabetes accounts for only approximately 10% in young Chinese diabetic population. There is increasing evidence that genetic factors interact with environmental and lifestyle factors, mainly reflected by obesity, to give rise to early-onset diabetes. As many as 50% of young Chinese diabetic patients have family history of diabetes. In a pilot study, the prevalence of mutations in glucokinase and HNF-1a genes in our young Chinese type 2 diabetic patients is 3% and 5%, respectively, while mutation in HNF-4a is uncommon (Chan, 2000; Ng et al., 1999). Hence, the cause of diabetes in the majority of young patients remains to be identified. Besides, 30– 50% of our young diabetic patients have significant albuminuria despite short disease duration of less than 5 years (Ko, Chan, Lau, & Cockram, 1999). End stage renal disease is the cause of death in 10 – 30% of Chinese type 2 diabetic patients (Colhoun et al., 2001). This is in contrast to Caucasians in whom ischaemic heart disease is the leading cause of death. Since genetic factors play an important role in the development of diabetic nephropathy (Rippin, Patel, & Bain, 2001), we hypothesized that HNF-1b mutation may be implicated in the development of young-onset diabetes with nephropathy. To test this hypothesis, we screened HNF-1b for mutations in a group of 74 Chinese unrelated subjects with familial early-onset type 2 diabetes who had albuminuria in their early course of disease.
2. Patients and methods A total of 74 unrelated Chinese subjects were studied. They presented with type 2 diabetes (1995 WHO criteria) before the age of 40 years and had at least one first degree relative affected with diabetes. A random spot urine sample followed by a timed collection (4- or 24-h) was used to document albuminuria. All of them had diabetic nephropathy (based on two out of three urine collections) defined as 24-h urinary albumin excretion (UAE) 300 mg/day, 4-h urinary albumin excretion rate (AER) 200mg/min, and/or spot urinary albumin – creatinine ratio (ACR) 25 mg/ mmol (Mogensen et al., 1995). Thirty-one percent of them met the minimal criteria for MODY (age of diagnosis before 25 years of age and presence of diabetes in two consecutive generations). These patients were selected from a Diabetes Registry containing detailed clinical and biochemical information of 2000 cases recruited between February 1995 and August 1997 from the Diabetes and Endocrine Center of the Prince of Wales Hospital, the teaching hospital of the Chinese University of Hong Kong.
The mean age of onset for the subjects (47% male) was 30 years (range 10 –40 years) and the mean age at the time of assessment was 40 years (range 22– 60 years). Fifty-three percent of patients were treated with insulin with or without oral drugs and 24.3% were on oral drugs only. The remaining 23% patients were on diet. The mean ( ± S.D.) HbAlc was 8.6 ± 2.1%. Their geometric mean plasma creatinine was 118.0mmol/l (range 35 – 1293mmol/l) and AER was 601.8 ± 3.46mg/min. Thirteen percent were treated with inhibitors of the renin – angiotensin – aldosterone system. Thirty-three percent of patients had clinical evidence of diabetic retinopathy and 6.4% had macrovascular complications, namely ischaemic heart disease, cerebrovascular accident, or significant peripheral vascular disease. The study was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong, and informed consent for DNA donation was obtained from each subject. 2.1. Screening for HNF-1 mutations The minimal promoter region, flanking introns and exons of the HNF-1b gene were screened for mutations by direct sequencing of polymerase chain reaction (PCR) products, as described previously (Horikawa et al., 1997). 2.2. Transfection studies The missence mutation was introduced into a DNA fragment, which was cloned into a plasmid, pcDNA3.1HNF-1b, encoding the type A form of human HNF-1b, as described previously (Lindner et al., 1999). The reporter gene construct, pGL3-RA, was prepared using methodology, as reported previously (Bjorkhaug et al., 2000). HeLa cells were grown and transfected with DNA either of the wild-type or mutant type of HNF-1b-containing plasmid, the reporter gene pGL3-RA, pRL-SV40 as control as well as pcDNA 3.1+ (Bjorkhaug et al., 2000; Lindner et al., 1999). The transactivation activity of the normal and mutant HNF1b proteins was measured (Bjorkhaug et al., 2000). 2.3. Clinical studies All patients underwent a structured assessment including documentation of family history, as well as clinical and biochemical characteristics. All patients had a random spot urine sample followed by a timed urinary collection, either 4- or 24-h, for measurement of UAE. For patients confirmed to have HNF-1b mutation, ultrasonographic examination of kidney was carried out to screen for renal cysts and other urogenital abnormalities, which have been reported to be associated with the mutations. Family members of probands identified with HNF-1b mutation, if available, were recruited. All of them underwent 75-g oral glucose tolerance test (OGTT) and ultrasonography. Direct sequencing to screen for mutations was performed in all family members who agreed to undergo assessment.
W.Y. So et al. / Journal of Diabetes and Its Complications 17 (2003) 369–373
371
Fig. 1. Pedigree of Hong Kong Chinese family HK34. The proband was labelled with arrow and other subjects studied are labelled (a) – (f ). Details are described in text. Subjects with diabetes are indicated by filled symbols. Genotype: N, normal allele; M, E260D allele. BMI: body mass index (kg/m2), ACR: albumin – creatinine ratio (mg/mmol), plasma creatinine in mmol/l.
Plasma glucose concentration was measured by a glucose oxidase method (Diagnostic Chemicals, Charlottetown, Prince Edward Island, Canada). HbAlc was measured by gel electrophoresis (Ciba Corning Diagnostic, Palo Alto, USA). Data are expressed as mean ± S.D. or geometric mean ± antilog S.D. as appropriate.
3. Results Of the 74 patients, a missense genetic variant in exon 3 (E260D, GAG ! GAC) was identified in a male patient (HK34) who was diagnosed to have diabetes at the age of 36 years. At the time of examination in 1995, his age was 49
years, with a body mass index (BMI) of 30.2 kg/m2 and blood pressure of 162/100 mm Hg. His 24-h UAE was 832.4 mg/day and plasma creatinine was 101mmol/l (normal range 44 –107mmol/l). Clinically, there was no evidence of other glomerulopathy. His HbAlc was 6.9% (normal range 5.1 – 6.4%) and there was no other diabetic complications including retinopathy. He was treated with oral agents. The same mutation was also present in 2 of 150 (1.3%) unrelated nondiabetic Chinese subjects who had normal renal function. Screening of the other family members of HK34 revealed the same variant in four of the siblings available, in whom three of them (aged 38 (subject c), 34 (subject e), and 32 (subject f) years, respectively) had normal glucose tolerance using the 1998 WHO criteria (Fig. 1). Subject c and f had
Fig. 2. Result of transfection study using transcriptional activation by epitope-tagged wild-type and E260D of HNF-1b. Details of method are described in text. There was no significant difference in activity between the wild-type and the E260D variant. The addition of the wild-type and E260D variant resulted in additional effect in a dose-dependent manner.
372
W.Y. So et al. / Journal of Diabetes and Its Complications 17 (2003) 369–373
normoalbuminuria and their BMI were 19.7 and 28.4 kg/m2, respectively. Subject e had microalbuminuria with a spot urine ACR of 5.0 mg/mmol (normal range < 3.5 mg/mmol), plasma creatinine of 63 mmol/l, and BMI of 20.8 kg/m2. One sibling (subject d) was found to have diabetes based on 75-g OGTT at the age of 36 years (plasma glucose at 0 and 120 min were 4.9 and 13.2 mmol/l, respectively). She had a BMI of 23.5 kg/m2 and had normoalbuminuria with plasma creatinine of 57mmol/l. The other sibling (subject b) had impaired glucose tolerance (IGT) (plasma glucose of 5.8 and 9.8 mmol/l at 0 and 120 min, respectively) at age of 40 years. He had a BMI of 26.2 kg/m2 and a plasma creatinine of 68 mmol/l. The proband’s mother had unknown status of glucose tolerance and she died at the age of 65 years due to heart disease. Ultrasonographic studies revealed multiple renal cysts and fatty liver in the proband. Renal cysts were also identified in subject f and there was no urogenital abnormalities identified in subjects c, d, and e (Fig. 1). Transfection studies revealed that there was insignificant difference for stimulated transcription of a firefly luciferase reporter gene linked to the HNF-1 binding site of the rat albumin gene (Fig. 2). Also, there was no inhibition of HNF-1b activity when cells were transfected with equivalent amounts of wild-type and mutant HNF-1b expression vectors, implying the mutation did not function in a dominant-negative manner. In addition, a polymorphism in exon 5 (Q378Q, CAG ! CAA) was identified in a 50-year-old patient who had diabetes since 37 years of age. He was treated with an oral agent. HbAlc was 9.4% and plasma creatinine was 105 mmol/l. The polymorphism Q378Q was not present in 105 unrelated nondiabetic Chinese subjects. There were no other mutations nor polymorphisms identified in the cohort.
4. Discussion According to the WHO Multinational Study of Vascular Disease in Diabetes findings, Chinese diabetic subjects have increased risk of developing renal disease. In contrast to Caucasian populations where cardiovascular disease is the leading cause of death, 30– 50% of Chinese diabetic patients die from end stage renal disease (Colhoun et al., 2001). Furthermore, 30– 50% of Chinese patients have albuminuria, which predicts the decline of renal function (Chan et al., 1995). Mutations in HNF-1b are associated with MODY5, which is associated with varying severity of glycaemia and renal failure. Apart from multiple renal cysts, which are usually present before the onset of diabetes (Horikawa et al., 1997; Lindner et al., 1999; Tomura et al., 1999), severe urogenital developmental abnormalities were identified in a Norwegian pedigree, suggesting a broad spectrum of clinical syndrome beyond glucose intolerance associated with this mutation (Lindner et al., 1999). Based on knockout mice experiment, HNF-1b, which is expressed
in renal tubules, appears to play an important role in its development (Barbacci et al., 1999; Bingham et al., 2001; Coffinier, Thepot, Babinet, Yaniv, & Barra, 1999). In light of the high prevalence of young-onset diabetes and relatively high prevalence of nephropathy (30%), we examined the potential impact of HNF-1b gene mutations or polymorphisms in our young diabetic patients with clinical evidence of renal disease. We hypothesize that the gene may be a factor in the development of diabetes and may increase the susceptibility of developing nephropathy. The novel missense variant HNF-1b E260D identified in our patient affects a highly conserved amino acid (human to Xenopus) in the DNA binding domain. Changing the amino acid from glutamic to aspartic acid results, however, in only a minor change in the carbon chain involved in protein – protein and protein – DNA interaction and with minimal changes in the protein charge. Besides, our transfection studies suggest only a minor effect of the variant on protein function. The proband carrying the mutation had macroalbuminuria with multiple renal cysts. Age of onset of diabetes was 36 years of age and he was treated with oral drugs. Two of the siblings (subjects d and b) were carriers of the mutation, with either diabetes (subject d) and impaired glucose tolerance (subject b). Both were obese according to the WHO Western Pacific Region criteria for Asian populations ( 25 kg/m2), but they had no renal or urogenital abnormalities in terms of albuminuria and on ultrasonographic imaging. The other siblings carrying the mutation had normal glucose tolerance although 2 of them had either albuminuria (subject e) or renal cysts on ultrasound imaging (subject f). Subject c, who had a BMI of 19.7 kg/m2, had normal glucose tolerance and normoalbuminuria. The heterogeneity of the phenotype in terms of glycaemia and urogenital manifestations together with results from the transfection studies suggested that E260D mutation might not play a major role in the development of diabetes or nephropathy. Nonetheless, it is plausible that it might interact with other modifying genes and/or risk factor such as raised blood pressure, increased plasma lipid or blood glucose to increase the risk of development of diabetes and its complications. To test this hypothesis, screening of the variant in a larger population will be required. Based on our pilot study, the frequency of HNF-1b variant in Chinese patients with familial early-onset type 2 diabetes was 1.4%. This finding is in agreement with the reported 2.5% prevalence in Japanese type 2 diabetic patients with early-onset disease (Horikawa et al., 1997). Given the low frequency of these genetic variants in both diabetic and nondiabetic subjects in different ethnic groups including Chinese, the heterogeneity of phenotypes in carriers and insignificant results from the transfection study, we concluded that HNF-1b genetic variants are not the major causes of early-onset type 2 diabetes or MODY but might have modifying effects. The clinical significance regarding its associations with renal manifestation in terms
W.Y. So et al. / Journal of Diabetes and Its Complications 17 (2003) 369–373
of either albuminuria or renal cysts also requires further elucidation (Beards et al., 1998; Weng et al., 2000).
References Barbacci, E., Reber, M., Ott, M. O., Breillat, C., Huetz, F., & Cereghini, S. (1999). Variant hepatocyte nuclear factor 1 is required for visceral endoderm specification. Development, 126, 4795 – 4805. Beards, F., Frayling, T., Bulman, M., Horikawa, Y., Allen, L., Appleton, M., Bell, G. I., Ellard, S., & Hattersley, A. T. (1998). Mutations in hepatocyte nuclear factor 1beta are not a common cause of maturity-onset diabetes of the young in the UK. Diabetes, 47, 1152 – 1154. Bingham, C., Bulman, M. P., Ellard, S., Allen, L. I., Lipkin, G. W., Hoff, W. G., Woolf, A. S., Rizzoni, G., Novelli, G., Nicholls, A. J., & Hattersley, A. T. (2001). Mutations in the hepatocyte nuclear factor1beta gene are associated with familial hypoplastic glomerulocystic kidney disease. American Journal of Human Genetics, 68, 219 – 224. Bingham, C., Ellard, S., Allen, L., Bulman, M., Shepherd, M., Frayling, T., Berry, P. J., Clark, P. M., Lindner, T., Bell, G. I., Ryffel, G. U., Nicholls, A. J., & Hattersley, A. T. (2000). Abnormal nephron development associated with a frameshift mutation in the transcription factor hepatocyte nuclear factor-1 beta. Kidney International, 57, 898 – 907. Bjorkhaug, L., Ye, H., Horikawa, Y., Sovik, O., Molven, A., & Njolstad, P. R. (2000). MODY associated with two novel hepatocyte nuclear factor-1alpha loss-of-function mutations (P112L and Q466X). Biochemical and Biophysical Research Communications, 279, 792 – 798. Cereghini, S., Ott, M. O., Power, S., & Maury, M. (1992). Expression patterns of vHNF1 and HNF1 homeoproteins in early postimplantation embryos suggest distinct and sequential developmental roles. Development, 116, 783 – 797. Chan, J. C. (2000). Heterogeneity of diabetes mellitus in the Hong Kong Chinese population. The Chinese University of Hong Kong-Prince of Wales Hospital Diabetes Research and Care Group. Hong Kong Medical Journal, 6, 77 – 84. Chan, J. C., Cheung, C. K., Cheung, M. Y., Swaminathan, R., Critchley, J. A., & Cockram, C. S. (1995). Abnormal albuminuria as a predictor of mortality and renal impairment in Chinese patients with NIDDM. Diabetes Care, 18, 1013 – 1016. Chan, J. C., Cheung, C. K., Swaminathan, R., Nicholls, M. G., & Cockram, C. S. (1993). Obesity, albuminuria and hypertension among Hong Kong Chinese with non-insulin-dependent diabetes mellitus (NIDDM). Postgraduate Medical Journal, 69, 204 – 210. Cockram, C. S., Woo, J., Lau, E., Chan, J. C., Chan, A. Y., Lau, J., Swaminathan, R., & Donnan, S. P. (1993). The prevalence of diabetes mellitus and impaired glucose tolerance among Hong Kong Chinese adults of working age. Diabetes Research and Clinical Practice, 21, 67 – 73. Coffinier, C., Thepot, D., Babinet, C., Yaniv, M., & Barra, J. (1999). Essential role for the homeoprotein vHNF1/HNF1beta in visceral endoderm differentiation. Development, 126, 4785 – 4794. Colhoun, H. M., Lee, E. T., Bennett, P. H., Lu, M., Keen, H., Wang, S. L.,
373
Stevens, L. K., & Fuller, J. H. (2001). Risk factors for renal failure: the WHO Mulinational Study of Vascular Disease in Diabetes. Diabetologia, 44 (Suppl. 2), S46 – S53. Fajans, S. S., Bell, G. I., & Polonsky, K. S. (2001). Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young. New England Journal of Medicine, 57, 971 – 980. Horikawa, Y., Iwasaki, N., Haram, N., Furuta, H., Hinokio, Y., Cockburn, B. N., Lindner, T., Yamagata, K., Ogara, M., Tomonaga, O., Kuroki, H., Kasahara, T., Iwamoto, Y., Bell, GI. (1997). Mutation in hepatocyte nuclear factor-1 beta gene (TCF2) associated with MODY. Nature Genetics, 17, 384 – 385. Janus, E. D., Watt, N. M., Lam, K. S., Cockram, C. S., Siu, S. T., Liu, L. J., & Lam, T. H. (2000). The prevalence of diabetes, association with cardiovascular risk factors and implications of diagnostic criteria (ADA 1997 and WHO 1998) in a 1996 community-based population study in Hong Kong Cardiovascular Risk Factor Steering Committee, American Diabetes Association. Diabetic Medicine, 17, 741 – 745. Ko, G. T., Chan, J. C., Lau, M., & Cockram, C. S. (1999). Diabetic microangiopathic complications in young Chinese diabetic patients: a clinicbased cross-sectional study. Journal of Diabetes and its Complications, 13, 300 – 306. Lindner, T. H., Njolstad, P. R., Horikawa, Y., Bostad, L., Bell, G. I., & Sovik, O. (1999). A novel syndrome of diabetes mellitus, renal dysfunction and genital malformation associated with a partial deletion of the pseudo-POU domain of hepatocyte nuclear factor-1beta. Human Molecular Genetics, 8, 2001 – 2008. Menzel, R., Kaisaki, P. J., Rjasanowski, I., Heinke, P., Kerner, W., & Menzel, S. (1998). A low renal threshold for glucose in diabetic patients with a mutation in the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene. Diabetic Medicine, 15, 816 – 820. Mogensen, C. E., Vestbo, E., Poulsen, P. L., Christiansen, C., Damsgaard, E. M., EiskjÆr, H., FrØland, A., Hansen, K. W., Nielsen, S., & Pedersen, M. M. (1995). Microalbuminuria and potential confounders. Diabetes Care, 18, 572 – 581. Ng, M. C., Cockburn, B. N., Lindner, T. H., Yeung, V. T., Chow, C. C., So, W. Y., Li, J. K., Lo, Y. M., Lee, Z. S., Cockram, C. S., Critchley, J. A., Bell, G. I., & Chan, J. C. (1999). Molecular genetics of diabetes mellitus in Chinese subjects: identification of mutations in glucokinase and hepatocyte nuclear factor-1alpha genes in patients with early-onset type 2 diabetes mellitus/MODY. Diabetic Medicine, 16, 956 – 963. Rey-Campos, J., Chouard, T., Yaniv, M., & Cereghini, S. (1991). vHNF1 is a homeoprotein that activates transcription and forms heterodimers with HNF1. EMBO Journal, 10, 1445 – 1457. Rippin, J. D., Patel, A., & Bain, S. C. (2001). Genetics of diabetic nephropathy. Best Practice Research and Clinical Endocrinology and Metabolism, 15, 345 – 358. Tomura, H., Nishigori, H., Sho, K., Yamagata, K., Inoue, I., & Takeda, J. (1999). Loss-of-function and dominant-negative mechanisms associated with hepatocyte nuclear factor-1beta mutations in familial type 2 diabetes mellitus. Journal of Biological Chemistry, 274, 12975 – 12978. Weng, J. P., Lehto, M., Forsblom, C., Huang, X., Li, H., & Groop, L. C. (2000). Hepatocyte nuclear factor-1 beta (MODY5) gene mutations in Scandinavian families with early-onset diabetes or kidney disease or both. Diabetologia, 43, 131 – 132.