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STUDY OF OCCURRENCE OF AMINO ACID MUTATION (Ala98Val) OF HNF1¡ IN ASSOCIATION WITH TYPE II DIABETES IN DIABETIC POPULATION OF KATHMANDU
Maturity onset diabetes of the young [MODY] type 3 is a monogenic form of diabetes. Gene defects in the Hepatocyte Nuclear Factor -1 alpha (HNF1¡) causes MODY3. HNF1¡ gene located in the chromosome (12q24.2) codes for a transcription factor which helps in signalling of insulin exocytosis in pancreatic Beta cells. A prevalent amino acid mutation at codon 98-Ala98Val (exon 1) of the HNF1¡ was shown to be associated with diabetes in the South Indian population. This study was designed to investigate the occurrence of amino acid polymorphism at codon 98- Ala98val of HNF1¡ in association to type II diabetic population of Kathmandu. DNA samples were randomly collected from 12 non-diabetic and 56 diabetic patients. The DNA samples were amplified using Polymerase Chain Reaction (PCR). Restriction Fragment Length Polymorphism (RFLP) was carried out to identify the occurrence of the mutation. During the study, 16.17% of Ala98Val mutation was observed among 68 samples.
A prevalent amino acid polymorphism at codon 98-Ala98Val (exon 1) of the HNF1¡ was shown to be associated with diabetes in the South Indian population.12 Since Nepal shares the ancestral origin with India and people have been sharing similar lifestyles for a long period of life it was relevant to check the occurrence of same mutation in diabetic population of Nepal as well.
The study was carried out to identify the occurence of amino acid mutation (Ala98Val) of HNF 1 alpha in association with type 2 diabetes in diabetic population of Kathmandu.
DNA samples were randomly collected from 12 non-diabetic and 56 diabetic patients. The DNA samples were amplified using Polymerase Chain Reaction (PCR). Restriction Fragment Length Polymorphism (RFLP) was carried out to identify the occurrence of the mutation.
During the study, 16.17% of Ala98Val mutation was observed among 68 samples.
The studies showed the occurrence of Ala98Val amino acid mutation in diabetic samples that were taken under study.
Diabetes, Type II Diabetes, Hepatocyte Nuclear Factor 1¡, Restriction Fragment Length Polymorphism, Single Nucleotide Polymorphism, Ala98Val
What this study adds:
1. What is known about this topic?
This study is first of its kind in Nepal, regarding polymorphism in HNF1¡ in diabetic population.
2. What new information is offered in this study?
The study showed the occurrence of Ala98Val amino acid mutation in diabetic patients of Kathmandu that were taken under study.
3. What are the implications for research, policy or practice?
The establishment of association and detection of polymorphism would definitely be useful in prognosis, susceptibility study and medication of the diabetes in future.
Hepatocyte Nuclear Factor-1alpha (HNF1¡) is a transcription factor found in pancreatic beta cells1 and hepatocytes2. It helps in regulation of differentiation of pancreatic beta cells3 and in transcription of genes required for insulin secretion.4 Mutation in HNF1¡ has been found to cause a type of monogenic diabetes called MODY3.5
Mutation in HNF1¡ gene, located on the chromosome 12 (12q24.2), is found to be associated with both late onset type II diabetes6 and type I diabetes.7,8,9 Variation in this gene has been reported in Finnish6, Danish Caucasians10, Chinese and Japanese subjects11 in association with type II diabetes and MODY. Mutation in this gene has been shown to cause type I diabetes in Japanese subjects.7,8,9
Mutational hotspot in exon 4 of HNF1¡ has been demonstrated by the studies in Germany12 and in Finnish and North American populations. 13 A prevalent amino acid polymorphism at codon 98-Ala98Val (exon 1) of the HNF1¡ was shown to be associated with diabetes in the South Indian population. 12
A particular polymorphism, ala98val, has been reported in Danish Caucasians and Finnish population.6,10 More recently, ala98val has been found to be prevalent in South Indian population as well.14 This study has thus been designed to detect the occurrence of ala98val polymorphism in people of Nepal.
68 blood samples were collected; 12 from non-diabetic subjects from SANN International College and 56 from Annapurna Neurological Hospital. Informed consent, made under proper format of Nepal Health Research Council (NHRC), was obtained from all the participants.
DNA was extracted from the blood samples by phenol chloroform DNA extraction method.15 HNF1¡ gene segment (251bp including region of mutation) was PCR amplified using primers described earlier.14 The sequences of the sense and antisense primers (Promega corporation) : 5-GAAGGCCCCCTGGACAAG G-3 and 5-CCCTCTAGGCTCTCCTGGGA-3 respectively.
The PCR (long-gene PCR machine) was carried out in a volume of 50ul containing: 1 ul DNA, 1.5 mmol/l MgCl2, 1mmol/l dNTPs, 5 pmol of each primer, and 1unit of Taq DNA polymerase (Promega corporation). The PCR conditions set were: denaturation (95° for 30 s), annealing (65° for 30 s), extension (72° for 30 s) followed by 35 cycles, and a final extension (72° for 9 min). 3 units of the enzyme HaeIII was used for 3hrs to carry out Restriction digestion. The cleaved products were run in 3% agarose gel containing ethidium bomide. The variation in digested fragments was visualized under UV gel-documentation system. The study was conducted in molecular biology laboratory of Department of Biotechnology, SANN International College.
68 samples were considered during the study, out of which 12 were non-diabetic and 56 were diabetic. Out of 12 non-diabetic subjects, 3 showed heterozygous ala/val polymorphism while none showed homozygous val/val polymorphism. Similarly, out of 56 diabetic subjects, 8 showed heterozygous ala/val polymorphism and 3 showed val/val homozygous polymorphism. Rest of the subjects did not show any polymorphism. In sum, 11 out of 68 subjects showed ala98val polymorphism which is 16.17%.
The study carried out by Anuradha et. al. showed the prevalence of ala98val polymorphism in association with MODY3 and early onset diabetes in South Indian population. This investigation on diabetic population of Kathmandu further reports the occurrence of ala98val polymorphism in relation to type II diabetic patients.
Similarly, the study showed presence of mutation in the youths of SANN College who showed no symptoms of diabetes. The presence of mutation in those samples shows the possibility of diabetes in near future and also indicates the need for genetic testing of their family members. Out of the 11 volunteers who had the mutation 5 of them have reported diabetes in their near relatives. This finding further supports the presence of mutation in the family. Since genetic testing of disease is a new practice in Nepal we suppose those volunteers might have running mutations in their family which they are unaware of.
The practice of testing plasma glucose for testing diabetes dates back to no more than roughly 20 years in Nepal. Therefore, the age of onset of the patients of diabetes can only be known from the last 20 years. Since MODY3 presents a mild form of diabetes people might be unaware of their exact age of higher plasma glucose. Furthermore due to practice of going for a check-up only after visible symptoms the exact age of onset is difficult to be known.
The presence of mutation in youths shows that there may be presence of slightly higher plasma glucose in them. It would be a good suggestion for them to go for routine check-up and correctly diagnose MODY3 and be sure it is not type I diabetes. The result may alter their medication process.
This study is first of its kind in Nepal, regarding polymorphism in HNF1¡ in diabetic population. The occurrence of the ala98val polymorphism could have biological significance, which remains to be established. The study on larger sample size with samples from different parts of Nepal would help to establish definite relation between ala98val polymorphism and diabetes in Nepalese population. The establishment of association and detection of polymorphism would definitely be useful in prognosis, susceptibility study and medication of the diabetes.
This study shows that the detection of HNF1a mutation can aid in the prognosis of MODY3 and early management of Diabetes in young people. However this is not the confirmatory diagnosis and other techniques like routine glucose tests should be performed to ensure the occurrence of Diabetes in such groups. Furthermore, new therapeutic approaches can be suggested such as use of sulphonylurea drugs in place of insulin for subjects with HNF1a mutations.
- Fajans SS, Bell GI, Polonsky KS. Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young. N Engl J Med. 2001;345(13):971-80. Epub 2001/09/29.
- Cereghini S. Liver-enriched transcription factors and hepatocyte differentiation. FASEB J. 1996;10(2):267-82. Epub 1996/02/01.
- Ryffel GU. Mutations in the human genes encoding the transcription factors of the hepatocyte nuclear factor (HNF)1 and HNF4 families: functional and pathological consequences. J Mol Endocrinol. 2001;27(1):11-29. Epub 2001/07/21.
- Dukes ID, Sreenan S, Roe MW, Levisetti M, Zhou YP, Ostrega D, et al. Defective pancreatic beta-cell glycolytic signaling in hepatocyte nuclear factor-1alpha-deficient mice. J Biol Chem. 1998;273(38):24457-64. Epub 1998/09/12.
- Yamagata K, Oda N, Kaisaki PJ, Menzel S, Furuta H, Vaxillaire M, et al. Mutations in the hepatocyte nuclear factor-1alpha gene in maturity-onset diabetes of the young (MODY3). Nature. 996;384(6608):455-8. Epub 1996/12/05.
- Rissanen J, Wang H, Miettinen R, Karkkainen P, Kekalainen P, Mykkanen L, et al. Variants in the hepatocyte nuclear factor-1alpha and -4alpha genes in Finnish and Chinese subjects with late-onset type 2 diabetes. Diabetes care. 2000;23(10):1533-8. Epub 2000/10/07.
- Yoshiuchi I, Yamagata K, Yoshimoto M, Zhu Q, Yang Q, Nammo T, et al. Analysis of a non-functional HNF-1alpha (TCF1) mutation in Japanese subjects with familial type 1 diabetes. Hum Mutat. 2001;18(4):345-51. Epub 2001/10/23.
- Yamada S, Nishigori H, Onda H, Utsugi T, Yanagawa T, Maruyama T, et al. Identification of mutations in the hepatocyte nuclear factor (HNF)-1 alpha gene in Japanese subjects with IDDM. Diabetes. 1997;46(10):1643-7. Epub 1997/10/06.
- Kawasaki E, Sera Y, Yamakawa K, Abe T, Ozaki M, Uotani S, et al. Identification and functional analysis of mutations in the hepatocyte nuclear factor-1alpha gene in anti-islet autoantibody-negative Japanese patients with type 1 diabetes. J Clin Endocrinol Metab. 2000;85(1):331-5. Epub 2000/01/14.
- Urhammer SA, Rasmussen SK, Kaisaki PJ, Oda N, Yamagata K, Moller AM, et al. Genetic variation in the hepatocyte nuclear factor-1 alpha gene in Danish Caucasians with late-onset NIDDM. Diabetologia. 1997;40(4):473-5. Epub 1997/04/01.
- Iwasaki N, Oda N, Ogata M, Hara M, Hinokio Y, Oda Y, et al. Mutations in the hepatocyte nuclear factor-1alpha/MODY3 gene in Japanese subjects with early- and late-onset NIDDM. Diabetes. 1997;46(9):1504-8. Epub 1997/09/01.
- Kaisaki PJ, Menzel S, Lindner T, Oda N, Rjasanowski I, Sahm J, et al. Mutations in the hepatocyte nuclear factor-1alpha gene in MODY and early-onset NIDDM: evidence for a mutational hotspot in exon 4. Diabetes. 1997;46(3):528-35. Epub 1997/03/01.
- Glucksmann MA, Lehto M, Tayber O, Scotti S, Berkemeier L, Pulido JC, et al. Novel mutations and a mutational hotspot in the MODY3 gene.Diabetes. 1997;46(6):1081-6. Epub 1997/06/01.
- Anuradha S, Radha V, Deepa R, Hansen T, Carstensen B, Pedersen O, et al. A prevalent amino acid polymorphism at codon 98 (Ala98Val) of the hepatocyte nuclear factor-1alpha is associated with maturity-onset diabetes of the young and younger age at onset of type 2 diabetes in Asian Indians. Diabetes care. 2005;28(10):2430-5. Epub 2005/09/28.
- Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2001. p. 6.8-6.11.
This work has been carried out in Molecular Biology laboratory of SANN International College, Department of Biotechnology. The authors would like to thank Ms. Sangya Poudyal and other laboratory staffs of SANN International College. The blood samples for the study were provided by Annapurna Neurological Institute and Allied Sciences.
Not commissioned. Externally peer reviewed
CONFLICTS OF INTEREST
The authors declare that they have no competing interests.
ETHICS COMMITTEE APPROVAL
Nepal Health Research Council, Reg. no. 151/2012