Method for the diagnosis of cell proliferative disease

Details Method for the diagnosis of cell proliferative disease Method for the diagnosis of cell proliferative disease

2001-03-15

2004-03-02

Souaya, Jehanne (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S091100, C536S023100, C536S024300

Reexamination Certificate

active

06699666

ABSTRACT:

TECHNICAL FIELD
This invention relates to a diagnostic method for detecting cell-proliferating diseases characterized by analyzing the methylation level of cytosine residues in the region involved in the expression of cytokine receptor gene.
BACKGROUND ART
DNA of eukaryotes happens to be methylated at cytosine residue of 5
th
position [Cell, 70, 5-8 (1992), Blood, 93, 4059-4070 (1999)]. It is known that the state of methylation of genome DNA of mammalians varies with differentiation or canceration of cells. Methylation reaction is catalyzed by enzymes, DNA(cytosine-5)methyltransferase (EC 2.1.1.37) [BioEssays, 17, 139-145 (1995)]. The enzyme methylates cytosine residue of dinucleotide sequence CpG or trinucleotide sequence CpNpG [N can be anyone of A (adenine), C (cytosine), G (guanine) and T (thymine)]. Furthermore, recognizing specifically the state where only one strand of double strand is methylated, the enzyme methylates cytosine residue in the complementary chain. Recently the existence of an enzyme catalyzing de-methylation of DNA was suggested [Proc. Natl. Acad. Sci. USA, 96, 5894-5896 (1999)]. It is known that the state of methylation of DNA happens to be transmitted to progeny through reproduction (meiosis) by a mode of inheritance called imprinting [Trend in Genet. (TIG), 13, 323-329 (1997)].
Non-coding regions of some genes have a part called CpG island with an abundance of CpG sequence. The state of methylation of cytosine residue in CpG island affects the transcription/expression of the gene. Namely the less methylation results in the accelerated transcription of the gene and the more methylation results in the suppressed transcription [Trends in Genet., 13, 444-449 (1997)]. CpG island affecting gene expression frequently resides in the promoter region of the gene but a case was reported where it resided in the intron [Nature, 389, 745-749 (1997)]. As for the mechanism that DNA methylation in CpG island results in suppressed gene expression, the followings are known. Methylated CpG island is combined with a protein called MeCP2(methyl CpG binding repressor 2) and activates de-acetylation enzyme of histone. As a result neighboring chromatin structure changes into condensed form, which prohibits RNA polymerase or transcription factor from entering into promoter region, and, as a result, the transcription/expression is suppressed [J. Biochem., 125, 217-222 (1999)].
Among cell-proliferating diseases it is known that DNA methylation is involved in cancer development [Adv. Cancer Res., 72, 141-196 (1998)]. However most of the reports advocate that CpG island of a cancer suppression gene is methylated, the expression is lowered and, as a result, cells become cancerous. There has been no report that methylation of cytokine receptor gene is involved. It was also not known with cell-proliferating diseases other than cancers that methylation pattern of genome DNA is changed.
Methods to analyze the state of methylation of genome DNA are known, for example, a method using methylation sensitive restriction enzymes, a method using chemical modification by hydrazine, permanganic acids or sodium bisulfite, an immunological method using antibodies specific to methylated DNA [Nucleic Acids Res., 26, 225-2264 (1998)], and affinity column chromatography method using MBD (methyl-CpG binding domain) like MeCP2 and DGGE (denaturing gradient gel electrophoresis) method [Proc. Natl. Acad. Sci. USA, 96, 2913-2918 (1999)]. Among them the method using sodium bisulfite has been widely used [Proc. Natl. Acad. Sci. USA, 89, 1827-1831 (1992), Nucl. Acids Res., 22, 2990-2997 (1994)].
The method is based on the following principle.
When single strand DNA produced by alkali denaturation is treated by sodium bisulfite, cytosine residues are changed into uracil residues by deamination, while methylated cytosine residues remain intact. Then polymerase chain reaction (hereinafter “PCR”) is performed using thus treated DNA as template. The primer therefor is designed to correspond to the base sequence wherein cytosines in base sequence to be amplified are replaced by thymines. When amplification is performed using such primers, methylated cytosine residues in the original genome DNA are amplified as cytosines, while unmethylated cytosine residues are amplified as thymine residues. Thus methylation in the original genome DNA is detected by determining the base sequence of the PCR product.
Psoriasis is a chronic inflammatory skin disease and a cell-proliferating disease accompanying abnormal proliferation of epidermal cells. More than 2% of caucasian population contract it [The Lancet, 350, 349-353 (1997), The Lancet, 338, 227-230 (1991), The Lancet, 338, 231-234 (1991)]. They get it after becoming adult in most cases. The cause of psoriasis is yet to be solved. Although families with high incidence of psoriasis are known and there are reports suggesting the involvement of genetic factor, the cause itself is not yet known [Nature Getetics, 14, 231-233 (1996), Science, 264, 1141-1145 (1994), Arch. Dermatol., 130, 216-224 (1994)]. On the other hand there is a report that the expression of epidermal growth factor receptor (EGF-R) at keratinocyte is accelerated at the affected part [J. Dermatol. Sci., 16, 120-128 (1998)]. While the expression of EGF-R in keratinocyte is normally induced by stimulation with interleukin-6 (IL-6), EGF-R was expressed at keratinocyte of psoriasis irrrespective of existence or absence of IL-6 stimulation.
The promoter region of EGF-R gene contains an abundance of CpG sequence and it was shown that transcription factors bind to the region [J. Biol. Chem., 266, 1746-1753 (1991), J. Biol. Chem., 263, 5693-5699 (1988)].
The sequence of the promoter region of human EGF-R gene is disclosed in accession number M38425 of GenBank database. THE CpG sequence is especially abundant and the binding sequences of transcription actor are scattered in the region about 500 bases upstream from the translation initiation point (1114
th
) and the region about 800 bases downstream from the initiation point of the 1
st
intron in the base sequence [J. Biol. Chem., 266, 1746-1753 (1991), J. Biol. Chem., 263, 5693-5699 (1988)].
Conventional diagnosis of psoriasis has been mostly performed by long time observation by dermatologists according to the diagnostic standard (PASI: psoriasis area and severity index) described in Dermatologica, 157, 238-244 (1978), J. Dermatol. Sci., 16, 165-169 (1998) and the like. Such diagnosis needs experienced dermatologists, and long time and much work or observing tissue lesions and symptoms. Therefore a speedy, reliable and reproducible diagnostic method has been desired.
Chronic rheumatoid arthritis is one of systemic autoimmune diseases and a cell-proliferating disease accompanying abnormal proliferation and inflammation of arthrosynovial cedes [Nippon Rinsho, 57, 333-338 (1999)]. The cause of chronic rheumatoid arthritis is yet to be solved. Although families with high incidence of chronic rheumatoid arthritis are known and the correlation with genotype of human histocompatibility antigen gene HLA-DR4 has been suggested, the cause itself is not yet known. There is a report that the activity of epidermal growth factor-like receptor 2 (erbB2/HER2
eu) is increased at the affected part [Seminars in Arthritis & Rheumatism, 21, 317-329 (1992)].
The sequence of the promoter region of human epidermal growth factor-like receptor 2 (erbB2/HER2
eu) gene is disclosed in accession number Z13970 of GenBank database. The region contains an abundance of CpG sequence and transcription factor Spl is suggested to bind to the region [Mol. Cell. Biol., 7, 2597-2601 (1987), Proc. Natl. Acad. Sci. USA, 84, 4374-4378 (1987), J. Biol. Chem., 265, 4389-4393 (1990), Gene 136, 361-364 (1993), Cancer Res., 54, 4193-4199 (1994)].
Conventional diagnosis of chronic rheumatoid arthritis has been perfor

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