Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-05-25
2004-10-05
Helms, Larry R. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C436S503000, C436S504000, C436S064000, C536S024300
Reexamination Certificate
active
06800436
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method of diagnostic of the diseases caused by mutations in the LKB1 gene and to an diagnostic reagent and a therapeutic preparation for said diseases.
BACKGROUND ART
Peutz-Jeghers (PJ) syndrome [MIM 175200] is an autosomal dominantly inherited disorder characterized by melanotic pigmentation of lips, perioral and buccal regions and by benign, hamartomatous and adenomatous types of multiple gastrointestinal polyps. Patients with this syndrome are known to frequently develop benign or malignant neoplasms in the gastrointestinal tract, pancreas, ovaries, testis, breast and uterus. In particular, small benign tumors frequently occur in the ovary and are developed as multifocal, symmetrical germinal-cord structures with annular tubules. Then, they progress into granulosa cancer and result in ambisexual precocity in girls. It was found that multifocal germinal cord cancer in boys, though with low frequency, results in gynecomasty and feminization due to over-production of estrogen. Defects in the gene responsible for Peutz-Jeghers syndrome (PJ gene) appear to predispose to a wide spectrum of neoplastic diseases. In fact, 50% of the carriers with a defect in one of PJ allele are known to develop cancer by the age of 60 (Giardiello, F. M. et al. Increased risk of cancer in Peutz-Jeghers syndrome. N. Engl. J. Med. 316,1511-1514 (1987); Spigelman, A. D., Murday, V. & Phillips, R. K. Cancer and Peutz-Jeghers syndrome. Gut 30, 1588-1590 (1989). [MIM 175200]). It is believed that loss or inactivation of the PJ gene product results in disruption of the fundamental growth control mechanism within somatic cells that have potential high proliferative capacity, which triggers the growth of benign hamartomatous polyps some of which turn into malignant tumor cells after further genetic alteration.
Recently, it was reported that PJ gene was mapped to chromosome 19p13.3 by linkage analysis in 12 families of PJ syndrome with a multipoint lod score of 7.00 at the microsatellite genetic marker D19S886 (Hemminki, A. et al. Localization of a susceptibility locus for PJ syndrome to 19p using comparative genomic hybridization and targeted linkage analysis. Nat. Genet. 15 (1), 87-90 (1997)). A similar linkage between PJ gene and the genetic marker D19S886 was reported in a second study investigating five other families as well (with a multipoint lod score of 7.52) (Amos, C. I. et al. Fine mapping of a genetic locus for PJ syndrome on chromosome 19p. Cancer Res. 57, 3653-3656 (1997)). D19S565 was first known as a genetic marker proximal to the PJ gene, which causes recombination with the gene. Thereafter, the genetic marker D19S878 was found to be located more proximal to the gene. These two makers, therefore, were thought to define the proximal border of the PJ candidate region. In both linkage studies, no recombination was observed between the marker D19S886 and PJ gene, indicating that they are located in a narrow interval.
DISCLOSURE OF THE INVENTION
An objective of the present invention is to identify the gene responsible for Peutz-Jeghers syndrome and to provide a method for diagnosing diseases resulting from a mutation in this gene, a diagnostic reagent and a therapeutic preparation for the diseases.
To identifiy the gene for PJ syndrome, the present inventors made a continuous cosmid contig and a restriction map for the region extending from 1.5 Mb within chromosome 19p13.3 which includes the PJ gene. EST database search for the genes mapped in this region was then performed, and the precise locations of these genes were determined. The inventors evaluated biological information of a number of genes thus found and selected several potent candidates of the PJ gene. Mutation analysis of these candidate genes in DNAs from patients with PJ syndrome revealed that one of the candidate genes, “LKB1,” was specifically mutated in the patients with PJ syndrome. Thus, the inventors' intense investigations successfully identified the gene responsible for PJ syndrome for the first time. Based on this finding, the inventors found that diseases caused by mutations in the LKB1 gene can be diagnosed and treated by utilizing the LKB1 gene, primers and probes based on the sequence thereof, LKB1 protein and antibodies that bind to LKB1 protein.
As described above, the present invention relates to a method of diagnosing diseases caused by mutation in the LKB1 gene, the gene responsible for PJ syndrome, and to a diagnostic reagent and a therapeutic preparation for the diseases. More specifically, the present invention relates to:
(1) a primer DNA used for diagnosing a disease caused by mutation in the LKB1 gene, the primer DNA comprising a nucleotide sequence containing at least a portion of any one of the nucleotide sequences shown in SEQ ID NOs: 1 to 4;
(2) the primer DNA according to (1), wherein the primer DNA has a nucleotide sequence corresponding to any one of the nucleotide sequences shown in SEQ ID NOs: 7 to 30;
(3) the primer DNA according to (1) or (2), wherein the disease caused by mutation in the LKB1 gene is Peutz-Jeghers syndrome;
(4) a probe DNA used for diagnosing a disease caused by mutation in the LKB1 gene, the probe DNA comprising a nucleotide sequence containing at least a portion of any one of the nucleotide sequences shown in SEQ ID NOs: 1 to 4;
(5) the probe DNA according to (4), wherein the disease caused by mutation in the LKB1 gene is Peutz-Jeghers syndrome;
(6) a therapeutic preparation for a disease caused by mutation in the LKB1 gene, the preparation comprising the LKB1 gene as an active ingredient;
(7) a therapeutic preparation for a disease caused by mutation in the LKB1 gene, the preparation comprising the LKB1 protein an active ingredient;
(8) a therapeutic preparation for a disease caused by mutation in the LKB1 gene, the preparation comprising a compound that enhances the activity of LKB1 protein as an active ingredient;
(9) the therapeutic preparation according to (6) to (8), wherein the disease caused by mutation in the LKB1 gene is Peutz-Jeghers syndrome;
(10) a reagent for diagnostic of a disease caused by mutation in the LKB1 gene, the reagent comprising an antibody that binds to the LKB1 protein as an active ingredient;
(11) the reagent according to (10), wherein the disease caused by mutation in the LKB1 gene is Peutz-Jeghers syndrome;
(12) a method of diagnosing a disease caused by mutation in the LKB1 gene, the method comprising detecting mutation in the LKB1 gene;
(13) a method of diagnosing a disease caused by mutation in the LKB1 gene, the method comprising the steps of:
(a) preparing a DNA sample from a patient;
(b) amplifying the DNA using the primer DNA according to (1);
(c) cleaving the amplified DNA;
(d) fractionating the DNA fragments according to their size;
(e) hybridizing the probe DNA according to (4) with the fractionated DNA fragments; and
(f) comparing the size of the DNA fragment thus detected to that from a control of a healthy subject;
(14) a method of diagnosing a disease caused by mutation in the LKB1 gene, the method comprising the steps of:
(a) preparing a RNA sample from a patient;
(b) fractionating the RNA sample depending on its size;
(c) hybridizing the probe DNA according to (4) with the RNA thus fractionated;
(d) comparing the size of the RNA thus detected to that from a control of a healthy subject;
(15) a method of diagnosing a disease caused by mutation in the LKB1 gene, the method comprising the steps of:
(a) preparing a DNA sample from a patient;
(b) amplifying the DNA using the primer DNA according to (1)
(c) separating the amplified DNA into single stranded DNA;
(d) fractionating the separated single stranded DNA on a non-denatured gel;
(e) comparing the mobility of the single stranded DNA separated on the non-denatured gel to that of a control of a healthy subject;
(16) a method of diagnosing a disease caused by mutation in the LKB1 gene, the method comprising the steps of:
(a) preparing a DNA sample from a patient;
(b) amplifying the DNA using the primer DNA acc
Jenne Dieter E.
Nezu Jun-ichi
Chugai Seiyaku Kabushiki Kaisha
Fish & Richardson P.C.
Helms Larry R.
Yu Misook
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