Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Radical -xh acid – or anhydride – acid halide or salt thereof...
Reexamination Certificate
2000-06-01
2004-06-01
Myers, Carla J. (Department: 1634)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Radical -xh acid, or anhydride, acid halide or salt thereof...
C536S023200, C536S023500, C435S006120, C435S194000
Reexamination Certificate
active
06743823
ABSTRACT:
TECHNICAL FIELD
The present invention relates to isolated polynucleotide molecules useful for analyzing carbamyl phosphate synthetase I phenotypes, to peptides encoded by these molecules, and to the diagnostic and therapeutic uses thereof relating to a newly identified carbamyl phosphate synthetase I polymorphism. Among such uses are methods for determining the susceptibility of a subject to hyperammonemia, decreased production of arginine and to bone marrow transplant toxicity based on an analysis of a sample isolated from tissue biopsies from the subject.
Table of Abbreviations
ABG
arterial blood gas(es)
ALI
acute lung injury
ASO
allele-specific oligonucleotide
ATP
adenosine triphosphate
BCAA
branched chain amino acid(s)
BMT
bone marrow transplant
BSA
bovine serum albumin
BuCy
busulfan, cyclophosphamide
BUN
blood urea nitrogen
CBVP16
cyclophosphamide, bis-
chloroethylnitrosourea, etoposide
cc
cubic centimeters
CPSI
carbamyl phosphate synthetase I
CTC
cyclophosphamide, thiotepa,
carboplatin
CVP16TBI
cyclophosphamide, etoposide, total
body irradiation
ECMO
extracorpreal membrane
oxygenation
fl
full length
GSHosc
glutathione synthetase
HAT
hypoxanthine, aminopterin, thymidine
HVOD
hepatic veno-occlusive disease
iNO
inhaled nitric oxide
KDa
kilodalton
KLH
keyhole limpet hemocyanin
I
liter
LAT
ligation activated translation
LCR
ligase chain reaction
MAS
meconium aspiration syndrome
NAG
n-acetyl glutamate
NASDA ™
nucleic acid sequence-based
amplification
NO or NO
x
nitric oxide
NOS
nitric oxide synthetase
O/C
omithine/citrulline
PBSCT
peripheral blood stem-cell
transplantation
PPHN
persistant pulmonary hypertension in
newborns
PCR
polymerase chain reaction
RCR
repair chain reaction
RDS
respiratory distress syndrome
REF
restriction endonuclease finger
printing
RT
reverse transcriptase
SSCP
single strand conformation
polymorphism
SDA
strand displacement activation
SNP
single nucleotide polymorphism
TC
thiotepa, cyclophosphamide
TEAA
total essential amino adds
UC
urea cycle
UCF
urea cycle function
VPA
valproic acid
BACKGROUND ART
The in vivo synthetic pathway for arginine commences with ornithine. Omithine is combined with carbamyl phosphate to produce citrulline, which in turn is combined with aspartate, in the presence of adenosine triphosphate (ATP), to produce argininosuccinate. In the final step, fumarate is split from argininosuccinate, to produce arginine. The degradative pathway for arginine is by the hydrolytic action of arginase, to produce omithine and urea. These reactions form the urea cycle. The urea cycle serves as the primary pathway for removing waste nitrogen produced by the metabolism of endogenous and exogenous proteins, and is shown schematically in FIG.
1
.
Disruption of metabolic processes is a frequent side effect of chemotherapy. Indeed, the agents used in high-dose chemotherapy affect a number of cellular processes. Metabolic processes localized in chemo-sensitive tissues, such as the liver and gastrointestinal tract, face a particularly great risk to disruption.
The constant turn-over and processing of nitrogen involves all the tissues in the body, but the first critical steps of the urea cycle are limited to the liver and gut. The high-dose chemotherapy associated with bone marrow transplant (BMT) interferes with liver function and is toxic to the intestine. Idiopathic hyperammonemia, which is suggestive of urea cycle dysfunction, has been reported to be associated with high mortality in patients undergoing bone marrow transplant. Davies et al.,
Bone Marrow Transplantation
, 17:1119-1125 (1996); Tse et al.,
American Journal of Hematology
, 38:140-141 (1991); and Mitchell et al.,
American Journal of Medicine
, 85:662-667 (1988).
A common complication of BMT is hepatic veno-occlusive disease (HVOD). HVOD is associated with jaundice, increased liver size and disruption of normal hepatic blood flow. HVOD occurs in approximately 20 to 40% of patients and is associated with severe morbidity and mortality.
Nitric oxide (NO) plays a role in regulating vascular tone and in maintaining patency of hepatic and pulmonary venules following high-dose chemotherapy. Intact urea cycle function is important not only for excretion of ammonia but in maintaining adequate tissue levels of arginine, the precursor of NO.
Carbamyl phosphate synthetase I (CPSI) is the rate limiting enzyme catalyzing the first committed step of ureagenesis via the urea cycle. CPSI is highly tissue specific, with function and production substantially limited to liver and intestines. Genomically encoded, CPSI is produced in the cytoplasm and transported into the mitochondria where it is cleaved into its mature 160 kDA monomeric form. The enzyme combines ammonia and bicarbonate to form carbamyl with the expenditure of two ATP molecules and using the co-factor N-acetyl-glutamate (NAG).
Any genetic predisposition to decreased urea cycle function would lead to hyperammonemia and would likely contribute to the severity of disorders associated with sub-optimal urea cycle function, including BMT-related toxicity. Thus, there is a need in the art for characterization of alleles present in populations suffering from disorders associated with suboptimal urea cycle function, undergoing BMT or otherwise facing exposure to environmental or pharmacological hepatotoxins. In view of the role of CPSI in the urea cycle, there is a particular need for characterization of CPSI alleles present in such populations.
SUMMARY OF THE INVENTION
A method of screening for susceptibility to sub-optimal urea cycle function in a subject is disclosed. The method comprising the steps of: (a) obtaining a nucleic acid sample from the subject; and (b) detecting a polymorphism of a carbamyl phosphate synthase I (CPSI) gene in the nucleic acid sample from the subject, the presence of the polymorphism indicating that the susceptibility of the subject to sub-optimal urea cycle function. In accordance with the present invention, detection of the polymorphism is particularly provided with respect to determining the susceptibility of a subject to bone marrow transplant toxicity.
Preferably, the polymorphism of the carbamyl phosphate synthetase polypeptide comprises a C to A transversion in exon 36 of the CPSI gene, more preferably at nucleotide 4340 of a cDNA that corresponds to the CPSI gene. More preferably, the C to A transversion at nucleotide 4340 of the cDNA that corresponds to the CPSI gene further comprises a change in the triplet code from AAC to ACC, which encodes a CPSI polypeptide having an threonine moiety at amino acid 1405.
The present invention also provides an isolated and purified biologically active CPSI polypeptide. Preferably, a polypeptide of the invention is a recombinant polypeptide. More preferably, a polypeptide of the present invention comprises human CPSI having an asparagine moiety at amino acid 1405.
The present invention also provides an isolated and purified polynucleotide that encodes a biologically active CPSI polypeptide. In a preferred embodiment, a polynucleotide of the present invention comprises a DNA molecule from a human. More preferably, a polynucleotide of the present invention comprises a cDNA that corresponds to the CPSI gene and which includes a C to A transversion at nucleotide 4340. Even more preferably, a polynucleotide of the present invention further comprises a cDNA that corresponds to the CPSI gene that includes a change in the triplet code from ACC to AAC at nucleotide 4340, and encodes a CPSI polypeptide having an asparagine moiety at amino acid 1405.
Kits and reagents, including oligonucleotides, nucleic acid probes and antibodies suitable for use in carrying out the methods of the present invention and for use in detecting the polypeptides and polynucleotides of the present invention are also disclosed herein. Methods for preparing the polynucleotides and polypeptides of the present invention are also disclosed herein.
In a further embodiment, this invention pertains to therapeutic methods based upon a polymorphism of a carbamyl phosphate synthase I (CPSI) gene as described her
Christman Brian W.
Summar Marshall L.
Jenkins & Wilson & Taylor, P.A.
Johannsen Diana
Myers Carla J.
Vanderbilt University
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