# Patent

## Title
HNF-1α gene including novel single-nucleotide polymorphism, protein encoded by the HNF-1α gene, and polynucleotide, microarray, kit, and method for diagnosis of MODY3

## Abstract

Provided are a HNF-1 α gene including a novel single-nucleotide polymorphism and a protein encoded by the HNF-1 α gene, a polynucleotide associated with MODY3 diabetes based on the HNF-1 α gene, a microarray and a diagnostic kit including the polynucleotide, and a method for diagnosis of MODY3 diabetes.

## Background
This application claims priority from Korean Patent Application No. 10-2004-0009626, filed on Feb. 13, 2004, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an HNF-1 α gene including a novel single-nucleotide polymorphism and a protein encoded by the HNF-1 α gene, a polynucleotide associated with MODY3 diabetes based on the HNF-1 α gene, a microarray and diagnostic kit including the polynucleotide, and a method for diagnosis of MODY3 diabetes.
2. Description of the Related Art
The genomes of all organisms undergo spontaneous mutation in the course of their continuing evolution, generating variant forms of progenitor nucleic acid sequences [Gusella, Ann. Rev. Biochem. 55, 831-854 (1986)]. The variant forms may confer an evolutionary advantage or disadvantage, relative to a progenitor form, or may be neutral. In some instances, a variant form confers a lethal disadvantage and is not transmitted to subsequent generations of the organism. In other instances, a variant form confers an evolutionary advantage to the species and is eventually incorporated into the DNA of most members of the species and effectively becomes the progenitor form. In many instances, both progenitor and variant form(s) survive and co-exist in a species population. The coexistence of multiple forms of a sequence gives rise to polymorphisms.
Several different types of polymorphisms have been known, including restriction fragment length polymorphisms (RFLPs), short tandem repeats (STRs), variable number tandem repeats (VNTRs), and single-nucleotide polymorphisms (SNPs). Among them, SNPs take the form of single-nucleotide variations between individuals of the same species. When SNPs occur in protein coding sequences, any one of the polymorphic forms may give rise to the expression of a defective or a variant protein. On the other hand, when SNPs occur in non-coding sequences, some of these polymorphisms may result in the expression of defective or variant proteins (e.g., as a result of defective splicing). Other SNPs have no phenotypic effects.
It is known that human SNPs occur at a frequency of 1 in about 1,000 bp. When such SNPs induce a phenotypic expression such as a disease, polynucleotides containing the SNPs can be used as primers or probes for diagnosis of a disease. Monoclonal antibodies specifically binding with the SNPs can also be used in diagnosis of a disease. Such SNPs can be detected by an analytic method such as polymerase chain reaction (PCR), sequencing, hybridization, and single-strand conformation polymorphism (SSCP).
It is known that 90-95% of total diabetes patients suffer type II diabetes mellitus. Type II diabetes mellitus is a disorder which is developed in persons who abnormally produce insulin or have low sensitivity to insulin, thereby resulting in large change in blood glucose level. When disorder of insulin secretion leads to the condition of type II diabetes mellitus, blood glucose cannot be transferred to body cells, which renders the conversion of food into energy difficult. It is known that a genetic cause has a role in type II diabetes mellitus. Other risk factors of type II diabetes mellitus are age over 45, familial history of diabetes mellitus, obesity, hypertension, and high cholesterol level. Currently, diagnosis of diabetes mellitus is mainly made by measuring a pathological phenotypic change, i.e., blood glucose level, using fasting blood glucose (FSB) test, oral glucose tolerance test (OGTT), and the like. When diagnosis of type II diabetes mellitus is made, type II diabetes mellitus can be prevented or its onset can be delayed by exercise, special diet, body weight control, drug therapy, and the like. In this regard, it can be said that type II diabetes mellitus is a disease in which early diagnosis is highly desirable.
MODY3 is a type of the maturity-onset diabetes of the young which is one of type II diabetes mellitus. It is estimated that MODY3 is responsible for 10-30% of type II diabetes mellitus. It is known that MODY3 is caused by disorder of HNF-1 α gene (also called as “MODY3 gene”). Millenium Pharmaceuticals Inc. reported that diagnosis and prognosis of type II diabetes mellitus can be made based on genotypic variations present on HNF1 gene [PR newswire, Sep. 1, 1998]. Even though there are reports about some genes associated with type II diabetes mellitus, researches into the incidence of type II diabetes mellitus have been focused on specific genes of some chromosomes in specific populations. For this reason, research results may vary according to human species. Furthermore, all causative genes responsible for MODY3 diabetes have not yet been identified. Diagnosis of MODY3 diabetes by such a molecular biological technique is now uncommon. In addition, early diagnosis before incidence of MODY3 diabetes is currently unavailable. Therefore, there is an increasing need to find new SNPs highly associated with MODY3 diabetes and related genes that are found in whole human genomes and to make early diagnosis of MODY3 diabetes using the SNPs and the related genes.
SUMMARY OF THE INVENTION
The present invention provides a polynucleotide useful for diagnosis or treatment of MODY3 diabetes.
The present invention also provides a microarray and kit useful for diagnosis of MODY3 diabetes.
The present invention also provides a method for efficient diagnosis of MODY3 diabetes.
The present invention also provides a variant HNF-1 α gene including a single-nucleotide polymorphism and a protein encoded by the variant HNF-1 α gene.
According to an aspect of the present invention, there is provided a polynucleotide for diagnosis or treatment of type II diabetes mellitus, including at least 10 contiguous nucleotides of a nucleotide sequence as set forth in SEQ ID NO: 3 and including a nucleotide at position 102 of the nucleotide sequence, or a complementary polynucleotide thereof.
According to another aspect of the present invention, there is provided a polynucleotide for diagnosis or treatment of type II diabetes mellitus, including at least 10 contiguous nucleotides of a nucleotide sequence as set forth in SEQ ID NO: 4 and including nucleotides at positions 102 and 103 of the nucleotide sequence, or a complementary polynucleotide thereof.
According to still another aspect of the present invention, there is provided a microarray for diagnosis of type II diabetes mellitus, including a substrate on which one of the polynucleotides of the present invention or the complementary polynucleotide thereof is immobilized.
According to still another aspect of the present invention, there is provided a kit for diagnosis of type II diabetes mellitus, including one of the polynucleotides of the present invention or the complementary polynucleotide thereof.
According to still another aspect of the present invention, there is provided a method for diagnosis of type II diabetes mellitus, which includes isolating a nucleic acid sample from an individual and determining a nucleotide sequence of a polymorphic site within a human HNF-1 α gene or a complementary polynucleotide thereof.
According to yet another aspect of the present invention, there is provided a variant HNF-1 α gene having a nucleotide sequence as set forth in SEQ ID NO: 1 and resulting from deletion of a cytidine nucleotide at position 1204 of a wild-type HNF-1 α gene (NCBI accession No. NM-000545).
According to a further aspect of the present invention, there is provided a variant HNF-1 α protein having an amino acid sequence as set forth in SEQ ID NO: 2.



BRIEF DESCRIPTION OF THE DRAWINGS
The above and other features and advantages of the present invention will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawing in which:
FIG. 1 is a view that illustrates the position of a polymorphic site of single-nucleotide polymorphism (SNP) P394fsdelC and a portion of a variant HNF-1 α protein encoded by the SNP.



DETAILED DESCRIPTION OF THE INVENTION
The present inventors found a single-nucleotide polymorphism (SNP) specific to MODY3 patients. That is, the present inventors found a HNF-1 α gene variant that is found in MODY3 patients but not in normal persons.
Therefore, the present invention provides a polynucleotide for diagnosis or treatment of MODY3 diabetes, including at least 10 contiguous nucleotides of a nucleotide sequence as set forth in SEQ ID NO: 3 and including a nucleotide at position 102 of the nucleotide sequence, or a complementary polynucleotide thereof.
The nucleotide sequence of SEQ ID NO: 3 is a nucleotide sequence of exon 6 of a wild-type HNF-1 α gene. The present inventors found that a cytidine nucleotide at position 102 of the nucleotide sequence of SEQ ID NO: 3 is specifically absent in a MODY3 diabetes patient group. That is, wild-type and variant HNF-1 α genes are different in a nucleotide of the position 102 (called as “polymorphic site”). In this regard, a likelihood of being diagnosed as at risk of developing MODY3 diabetes can be determined by determining a nucleotide at position 102 of exon 6 of a wild-type HNF-1 α gene.
The present invention also provides a polynucleotide for diagnosis or treatment of MODY3 diabetes, including at least 10 contiguous nucleotides of a nucleotide sequence as set forth in SEQ ID NO: 4 and including nucleotides at positions 102 and 103 of the nucleotide sequence, or a complementary polynucleotide thereof.
The nucleotide sequence of SEQ ID NO: 4 is a nucleotide sequence of exon 6 of a variant HNF-1 α gene. The nucleotide sequence of SEQ ID NO: 4 results from deletion of the cytidine nucleotide of the position 102 of the nucleotide sequence of SEQ ID NO: 3 and is the same as that of SEQ ID NO: 3 except the cytidine deletion. The nucleotide sequence of SEQ ID NO: 4 is specifically found in a MODY3 diabetes patient group. Therefore, the polynucleotide including the nucleotides of the positions 102 and 103 of SEQ ID NO: 4 and the complementary polynucleotide thereof cannot be completely hybridized with the nucleotide sequence of SEQ ID NO: 3 which is a wild-type sequence. In this respect, a likelihood of being diagnosed as at risk of developing MODY3 diabetes can be determined by determining the nucleotides of the positions 102 and 103 of SEQ ID NO: 4 or the nucleotide of the position 102 of SEQ ID NO: 3. The polynucleotide for diagnosis or treatment of MODY3 diabetes including both the nucleotides of the positions 102 and 103 of SEQ ID NO: 4 according to the present invention or the complementary polynucleotide thereof can be efficiently used in determining the nucleotides of the positions 102 and 103 of SEQ ID NO: 4 or the nucleotide of the position 102 of SEQ ID NO: 3 of an individual.
The polynucleotides of the present invention are used for specifically determining the SNP site newly found by the present inventors and each has a polymorphic site. In this regard, the polynucleotides of the present invention can be efficiently used in a method and an apparatus for diagnosis or treatment of MODY3 diabetes for a nucleic acid sample obtained from an individual. For example, in a case where the polynucleotides of the present invention are used as primers for PCR, each of the polynucleotides of the present invention can amplify one of a wild-type nucleotide sequence and a variant nucleotide sequence since it includes a wild-type or variant nucleotide in the polymorphic site. Whether a genomic DNA or mRNA of an individual is a wild-type or a variant can be determined by comparing primers used and PCR products. Therefore, it can be determined whether the individual has a higher likelihood of being diagnosed as at risk of developing MODY3 diabetes. In an exemplary embodiment of the present invention, the polynucleotides of the present invention are used as allele-specific primers. The polynucleotides of the present invention can also be used as allele-specific probes in an analytic method such as southern blot, as previously described in terms of the primers for PCR.
Therefore, in an exemplary embodiment of the present invention, the polynucleotides of the present invention are primer or probe polynucleotides. Preferably, the polynucleotides of the present invention have 10 or more nucleotides. The polynucleotides of the present invention are 10 to 400 nucleotides in length, preferably 10 to 100 nucleotides in length, and more preferably 10 to 50 nucleotides in length.
In the present invention, the term “primer” refers to a single stranded oligonucleotide that acts as a starting point of template-directed DNA synthesis under appropriate conditions, for example in a buffer containing four different nucleoside triphosphates and polymerase such as DNA or RNA polymerase or reverse transcriptase, and at an appropriate temperature. The appropriate length of the primer may vary according to the purpose of use, generally 15 to 30 nucleotides. Generally, a shorter primer molecule requires a lower temperature to form a stable hybrid with a template. A primer sequence is not necessarily completely complementary with a template but must be complementary enough to hybridize with the template. Preferably, the 3′ end of the primer is aligned with the polymorphic site of SEQ ID NO: 3 or 4, i.e., the nucleotide at the position 102 of SEQ ID NO: 3 or the nucleotides at the positions 102 and 103 of SEQ ID NO: 4. The primer is hybridized with a target DNA containing a polymorphic site and starts an allelic amplification in which the primer exhibits complete homology with the target DNA. The primer is used in pair with a second primer hybridizing with an opposite strand. Amplified products are obtained by amplification using the two primers, which means that there is a specific allelic form. The primer of the present invention includes a polynucleotide fragment used in a ligase chain reaction (LCR).
In the present invention, the term “probe” refers to a hybridization probe, that is, an oligonucleotide capable of sequence-specifically binding with a complementary strand of a nucleic acid. Such a probe includes a peptide nucleic acid as disclosed in Science 254, 1497-1500 (1991) by Nielsen et al. The probe according to the present invention is an allele-specific probe. In this regard, when there are polymorphic sites in nucleic acid fragments derived from two members of the same species, the probe is hybridized with DNA fragments derived from one member but is not hybridized with DNA fragments derived from the other member. In this case, hybridization conditions should be stringent enough to allow hybridization with only one allele by significant difference in hybridization strength between alleles. Preferably, the central portion of the probe of the present invention, that is, position 7 for a 15 nucleotide probe, or position 8 or 9 for a 16 nucleotide probe, is aligned with the polymorphic site of SEQ ID NO: 3 or 4. Therefore, there can be caused a significant difference in hybridization between alleles. The probe of the present invention can be used in diagnostic methods for detecting alleles. The diagnostic methods include nucleic acid hybridization-based detection methods, e.g., southern blot. In a case where DNA chips are used for the nucleic acid hybridization-based detection methods, the probe may be provided as an immobilized form on a substrate of a DNA chip.
The present invention also provides a microarray for diagnosis of MODY3 diabetes, including a substrate on which one of the polynucleotides of the present invention or the complementary polynucleotide thereof is immobilized.
As used herein, the term “polynucleotide microarray” indicates an analysis system in which polynucleotide groups are immobilized in a high density on a substrate, in detail, a microarray in which each of the polynucleotide groups is immobilized on a predetermined region. Such a microarray is well known in the pertinent art. Examples of the microarray are disclosed in U.S. Pat. Nos. 5,445,934 and 5,744,305. A fabrication method for the polynucleotide microarray is illustrated in U.S. Pat. Nos. 5,744,305, 5,143,854, and 5,424,186. The above patent documents about polynucleotide microarrays and fabrication methods thereof are incorporated herein in their entireties by reference.
The present invention also provides a kit for diagnosis of MODY3 diabetes, including one of the polynucleotides of the present invention or the complementary polynucleotide thereof. The kit may include reagents necessary for polymerization, for example dNTPs, various polymerases, and a colorant, in addition to the polynucleotides of the present invention.
The present invention also provides a method for diagnosis of MODY3 diabetes, which includes isolating a nucleic acid sample from an individual and determining a nucleotide sequence of a polymorphic site within a human HNF-1 α gene or a complementary polynucleotide thereof.
The operation of isolating the nucleic acid sample from an individual is well known in the pertinent art. For example, the nucleic acid sample can be obtained by amplifying a target nucleic acid by PCR followed by purification. In addition to PCR, there may be used LCR (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)), self-sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874 (1990)), or nucleic acid sequence based amplification (NASBA). The last two methods are related with isothermal reaction based on isothermal transcription and produce 30 or 100-fold RNA single strands and DNA double strands as amplification products.
The nucleic acid sample can also be obtained from human tissue or blood stream. The nucleic acid sample may not necessarily be a pure nucleic acid and may vary according to an analytic method. For example, in PCR or in situ hybridization, a tissue or blood stream itself may be used as the nucleic acid sample without the separation of a nucleic acid from the tissue or the blood stream. The nucleic acid sample may be a natural nucleic acid, including genomic DNA or RNA (including mRNA).
As used herein, the term “human HNF-1 α gene” is used as the meaning including both wild-type and variant HNF-1 α genes. That is, the presence of a SNP can be determined by determining a nucleotide of position 1204 which is a polymorphic site of a wild-type HNF-1 α gene (NCBI accession No. NM-000545) or determining nucleotides of positions 1203 and 1204 which is a polymorphic site of SEQ ID NO: 4, followed by comparison with the nucleotide sequence of the wild-type gene.
In the present invention, the determination of the nucleotide sequence of the polymorphic site of the human HNF-1 α gene can be carried out by using a method known in the pertinent art. Such a method may include sequencing, PCR, RT-PCR, and a hybridization-based method such as southern blot or northern blot, but is not limited thereto. In such a sequence determination, the polynucleotides of the present invention or the complementary polynucleotides thereof can be efficiently used as synthetic primers, PCR primers, or probes.
In the method of the present invention, whether an individual has a higher likelihood of being diagnosed as at risk of developing MODY3 can be determined, for example, by determining the nucleotide sequence of the human HNF-1 α gene of the nucleic acid sample isolated according to the above description or its fragment including the polymorphic site, followed by comparison with the nucleotide sequence of the polymorphic site of the wild-type human HNF-1 α gene. Preferably, when the HNF-1 α gene of the nucleic acid sample isolated from the individual includes a cytidine (C) deletion, relative to the polymorphic site of the wild type HNF-1 α gene, it is determined that the individual has a higher likelihood of being diagnosed as at risk of developing MODY3.
The present invention also provides a variant HNF-1 α gene having a nucleotide sequence as set forth in SEQ ID NO: 1 and resulting from deletion of a cytidine nucleotide at position 1204 of a wild-type HNF-1 α gene (NCBI accession No. NM-000545).
The present invention also provides a variant HNF-1 α protein having an amino acid sequence as set forth in SEQ ID NO: 2.
Hereinafter, the present invention will be described more specifically by Example. However, the following Example is provided only for illustration and thus the present invention is not limited thereto.
Example
In this Example, genomic DNAs were extracted from blood streams of a clinical MODY putative patient group, followed by amplification of HNF-1 α genes, sequencing, and comparison with the sequence of a wild-type gene. As a result, a SNP specific to the patient group was detected.
1. Amplification of 10 Exons of MODY3 Gene
Genomic DNAs were extracted from blood streams of MODY3 diabetes patients and normal persons. DNA extraction was performed according to a known extraction method (Molecular cloning: A Laboratory Manual, p 392, Sambrook, Fritsch and Maniatis, 2nd edition, Cold Spring Harbor Press, 1989) and the specification of a commercial kit (QIAGEN Blood Midi kit). Among extracted DNAs, only DNAs having a purity (A260/A280 nm) of at least 1.7 were used.
The 10 exons of the HNF-1 α gene was amplified by PCR using the extracted DNAs as templates and primers capable of amplifying the 10 exons of the HNF-1 α gene. In detail, the PCR was performed by a common PCR method using 1-100 μg of each of the genomic DNAs as a template, Taq polymerase, and primers presented in Table 1 below. The thermal cycles of the PCR were as follows: incubation at 95° C. for 5 minutes; 30 cycles at 95° C. for 30 seconds, at 64° C. for 15 seconds, and at 72° C. for 1 minute; incubation at 72° C. for 3 minutes and storage at 4° C. PCR products were used in sequencing after being purified by QIAquick kit.







TABLE 1







PCR primers used in amplification of MODY3 gene










Name
Sequence







Mody 3 promoter sense(T7)
SEQ ID NO: 5



Mody 3 promoter antisense(T3)
SEQ ID NO: 6



Mody 3 exon1 sense(T7)
SEQ ID NO: 7



Mody 3 exon1 antisense(T3)
SEQ ID NO: 8



Mody 3 exon2 sense(T7)
SEQ ID NO: 9



Mody 3 exon2 antisense(T3)
SEQ ID NO: 10



Mody 3 exon3 sense(T7)
SEQ ID NO: 11



Mody 3 exon3 antisense(T3)
SEQ ID NO: 12



Mody 3 exon4 sense(T7)
SEQ ID NO: 13



Mody 3 exon4 antisense(T3)
SEQ ID NO: 14



Mody 3 exon5 sense(T7)
SEQ ID NO: 15



Mody 3 exon5 antisense(T3)
SEQ ID NO: 16



Mody 3 exon6 sense(T7)
SEQ ID NO: 17



Mody 3 exon6 antisense(T3)
SEQ ID NO: 18



Mody 3 exon7 sense(T7)
SEQ ID NO: 19



Mody 3 exon7 antisense(T3)
SEQ ID NO: 20



Mody 3 exon8 & 9 sense(T7)
SEQ ID NO: 21



Mody 3 exon8 & 9 antisense(T3)
SEQ ID NO: 22



Mody 3 exon10 sense(T7)
SEQ ID NO: 23



Mody 3 exon10 antisense(T3)
SEQ ID NO: 24










2. Analysis of SNPs in Amplified Target DNAs
The 10 exons of the amplified HNF-1 α gene were sequenced. The sequencing was performed as follows: sequencing PCR was performed using primers for each exon according to the ABI BigDye terminator cycle sequencing ready reaction kit method. PCR products were precipitated by alcohol, suspended in formaldehyde, heated at 95° C. for 5 minutes, and directly placed on 4° C. ice. The sequencing of the 10 exons of the HNF-1 α gene thus obtained was performed in ABI PRISM 3700 Genetic Analyzer. The sequence analysis was performed using DNAstar program.
According to the analysis results, the MODY3 patient group carried a deletion of the cytidine nucleotide at position 1204 of the wild-type HNF-1 α gene (NCBI accession No. NM-000545). Such a deletion was observed at a frequency of 1 in 300 patients but was not observed in the normal persons. Such a SNP was designated as “P394fsdelC”. The SNP resulted in an upstream shift of a translation stop codon, relative to the wild-type HNF-1 α gene. These analysis results are shown in FIG. 1. FIG. 1 illustrates the position of a polymorphic site of the SNP, P394fsdelC, and a variant HNF-1 α protein encoded by the SNP.
A polynucleotides of the present invention can be used in diagnosis, treatment, and fingerprinting analysis of MODY3 diabetes.
A microarray and kit including the polynucleotide of the present invention can be used for efficient diagnosis of MODY3 diabetes.
A method for diagnosis of MODY3 diabetes of the present invention can efficiently detect the presence or a risk of MODY3 diabetes.
A variant HNF-1 α gene or variant protein of the present invention can be efficiently used in development of drugs for diagnosis or treatment of MODY3 diabetes.

## Claims

1. An isolated polynucleotide comprising at least 30 contiguous nucleotides of SEQ ID NO: 4, wherein the at least 30 contiguous nucleotides include the nucleotides at positions 102 and 103 of SEQ ID NO: 4, or the complement thereof.

2. An isolated variant HNF-1α gene consisting of SEQ ID NO: 1.

3. The isolated polynucleotide of claim 1, which is 30 to 100 nucleotides in length, or the complement thereof.

4. The isolated polynucleotide of claim 1, which is a primer or a probe.

5. A microarray comprising a substrate on which the isolated polynucleotide of claim 1 or the complement thereof is immobilized.

6. A kit comprising the isolated polynucleotide of claim 1 or the complement thereof.