Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-06-24
2001-08-14
Myers, Carla J. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S006120, C435S091200, C435S091500, C435S091510, C536S023500, C536S024310, C536S024330
Reexamination Certificate
active
06274310
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the fields of clinical diagnosis and of screening for risk of genetically-determined disorders relating to pancreatic disease.
BACKGROUND OF THE INVENTION
Pancreatic disorders, particularly diabetes mellitus, constitute a major public health problem, as they affect a sizeable proportion of the population and have a profound negative impact on the overall health and quality of life of those individuals so afflicted. While a variety of pharmaceutical compositions have been developed to compensate for reductions in insulin production brought about by pancreatic deficiency, modifications in lifestyle, such as exercise, adherence to a sensible diet, avoidance of tobacco and moderation of alcohol consumption, can do much to alleviate its effects; therefore, it is desirable to conduct genetic screening to identify individuals who are predisposed toward the development of diabetes and other pancreatic disorders while they are still asymptomatic.
Maturity onset (type II) diabetes mellitus is a highly prevalent disease caused by an imbalance between insulin production by the endocrine pancreatic &bgr;-cells and the insulin requirements of peripheral tissues. This results in hyperglycemia and secondary cardiovascular, renal, ocular, and neurological complications. Susceptibility to type II diabetes is generally believed to be inherited as a complex polygenic trait. However, a distinct subset of early onset type II diabetes (maturity onset diabetes of the young: MODY) is transmitted as an autosomal dominant monogenic disorder (Tattersall and Fajans, 1975,
Diabetes,
24: 44-53). The identification of genes implicated in MODY can be regarded as an effective strategy to gain insight into the molecular pathogenesis of the more common and complex late onset forms of type II diabetes mellitus. To date, three distinct MODY genetic loci have been identified (Froguel et al., 1992,
Nature,
356: 162-164), two of which correspond to transcription factors expressed in pancreatic &bgr;-cells [HNF1&agr; (MODY3) and HNF4&agr; (MODY1)]. MODY2 is caused by mutations in the glucokinase gene.
Insulin promoter factor-1 (IPF-1) is a transcription factor known to mediate glucose-responsive stimulation of insulin gene expression and is necessary for pancreas development. This homeodomain protein, also known as IDX-1, STF-1 and PDX-1, is critical for development of the pancreas in mice and is a key factor for the regulation of the insulin gene in the &bgr;-cells of the endocrine pancreas (Miller et al., 1994,
EMBO J.,
13: 1145-1156; Leonard et al., 1993,
Mol. Endocr.,
7: 1275-1283; Ohlsson et al., 1993,
EMBO J.
12: 4251-4259; Jonnson et al., 1994,
Nature,
371: 606-609; Peshavaria et al., 1994,
Mol. Endocr.
8: 806-816; Peers et al., 1994,
Mol. Endocr..
8: 1798-1806). Targeted disruption of the Ipf1 gene encoding IPF-1 in transgenic mice results in a failure of the pancreas to develop (pancreatic agenesis), although heterozygosity for this mutation and the wild-type allele has no apparent deleterious consequence (Jonnson, 1994, supra; Offield et al., 1996,
Development,
122: 983-995). An object of the present invention comprises a screening assay by which to assess a patient's risk of developing MODY4, to distinguish between MODY4 and other forms of MODY and to assist in determining the genetic basis for other pancreatic disorders that might result from IPF-1 deficiency.
SUMMARY OF THE INVENTION
The invention is based on the discovery that a mutation of the IPF1 gene (herein referred to as an “IPF1 mutation”), which encodes the insulin promoter factor-1 (IPF-1) transcription factor, is indicative of pancreatic disease, in particular of form early onset type II diabetes and of pancreatic agenesis.
By “IPF1 mutation”, we mean any defect in the IPF1 gene or regulatory regions (promoter, intron, splice sites) which leads to a loss of biological activity of the native IPF-1 protein or to a novel or altered IPF-1 protein function. Such activity includes, but is not limited to, the transcriptional activation of a gene whose regulatory sequences comprise an IPF-1 binding motif, as defined herein.
As used herein, “pancreatic disease” is defined as encompassing the absence, underdevelopment or maldevelopment of the pancreas, or loss or impairment of pancreatic function in terms of either the production, storage, stability or secretion of factors such as proteins, lipids, carbohydrates or other messenger molecules essential for the homeostatic regulation of pancreatic functions. Such factors might include hormones, enzymes, fatty acids, proteins or derivatized proteins such as lipoproteins, glycoproteins or phosphoproteins. Specifically, “pancreatic disease” is used to denote pancreatic agenesis or malformation, diabetes or any other manifestation of disease due to malfunctioning of the pancreas.
“Mature onset diabetes of the young” or “MODY” is a term which refers to a particular subset of cases of type II diabetes, namely those in which the patient becomes diabetic at an early age, typically before age 25, but in which fasting hyperglycemia, if present, can be regulated without insulin for at least two years following onset of clinical symptoms and in which genetic transmission of the disease is by an autosomal dominant mechanism.
“MODY4” refers to MODY that has an IPF1 mutation as its underlying genetic cause.
“IPF-1 binding motif” is defined as any gene regulatory sequence to which IPF-1 binds specifically in order to regulate transcription of the associated gene. Such sequences include, but are not limited to: FAR-FLAT (also called INS1-FLAT), the Far-linked AT-rich element of the rat insulin 1 gene (5′- GATCCTTCTTAATCTAATTACCCTAGGTCTAA-3′) [SEQ ID NO: 17]; SMS-TAAT1, a FLAT-like element 438 to 461 nucleotides upstream of the rat somatostatin gene (5′-GATCCCTGATTGCATATTAATTCTCAGATA-3′) [SEQ ID NO: 18]; SMS-TAAT2, a FLAT-like element 290 to 303 nucleotides upstream of the rat somatostatin gene (5′-GATCCGATCTCAGTAATTAATCATGCACCA-3′) [SEQ ID NO: 19]; SMS-UE-B, the B domain of the rat somatostatin upstream enhancer (5′-GATCCGCGAGGCTAATGGTGCGTAAAAGCACTGGTGA-3′) [SEQ ID NO: 20]; and SMS-PS, a transcriptional silencer element 219-233 nucleotides upstream of the rat somatostatin gene (5′-GATCCAGGCAAGATTATTTGGTCA-3′) [SEQ ID NO: 21].
The invention comprises a procedure for screening for pancreatic disease in a patient, comprising performing a detection step for a mutation in the gene encoding insulin promoter factor 1, wherein detection of a mutation is indicative of pancreatic disease.
In a preferred embodiment, the further step is carried out of obtaining a positive result in which said patient is homozygous for a mutation in IPF1, homozygosity being indicative of pancreatic agenesis.
It is preferable that a further step is carried out of obtaining a positive result in which said patient is heterozygous for a mutation in IPF1, heterozygosity being indicative of the presence of diabetes mellitus, more preferable that said diabetes mellitus is of the form early onset type II and most preferable that said diabetes mellitus of said form early onset type II is mature onset diabetes of the young (MODY).
Preferably, the mutation in IPF1 is a deletion of a single base pair, more preferably, the mutation results in a translational frame shift, and most preferably, the mutation is IPF1&Dgr;C (also denoted Pro63fs&Dgr;C or Pro63fsdelC).
A further object of the present invention encompasses a method for screening a patient for a mutation in the gene encoding insulin promoter factor 1 (IPF-1), comprising the steps of providing a nucleic acid sample from said patient and detecting a mutation in said gene in said sample, wherein detection of a mutation is indicative of pancreatic disease.
It is contemplated that the family of said patient comprises individuals affected with pancreatic disease, that said patient is asymptomatic with regard to pancreatic dis
Habener Joel F.
Stoffers Doris A.
Johannsen Diana
Myers Carla J.
Palmer & Dodge LLP
The General Hospital Corporation
Williams Kathleen M.
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