Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-10-04
2002-10-01
Myers, Carla J. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023500, C536S024310, C536S024330
Reexamination Certificate
active
06458542
ABSTRACT:
TECHNICAL FIELD
The present invention relates to isolated polynucleotide molecules useful for analyzing cardiac potassium channel minK subunit variants, and to screening and diagnostic uses thereof relating to a polymorphism in the KCNE1 gene encoding the cardiac potassium channel minK subunit polypeptide. Among such uses are methods for determining the susceptibility of a subject to drug-induced cardiac arrhythmias based on an analysis of a biological sample isolated from the subject.
Table of Abbreviations
ANOVA
analysis of variance
APD
action potential duration
Asn or N
asparagine
ASO
allele-specific oligonucleotide
Asp or D
aspartate
ATP
adenosine triphosphate
BSA
bovine serum albumin
CI
confidence interval
CL
cycle length
KCNE1
gene encoding cardiac potassium
channel minK subunit polypeptide
KCNQ1
cardiac potassium channel gene
fl
full length
EAD
early after depolarization(s)
GSHosc
glutathione synthetase
HAT
hypoxanthine, aminopterin, thymidine
HERG
cardiac potassium channel gene
I
Kr
rapid cardiac delayed rectifier
potassium current
I
Ks
slow cardiac delayed rectifier
potassium current
KDa
kilodalton
KLH
keyhole limpet hemocyanin
L
liter(s)
LAT
ligation activated translation
LCR
ligase chain reaction
LQTS
long QT syndrome
minK
cardiac potassium channel subunit
polypeptide encoded by KCNE1
msec
millisecond(s)
NAG
n-acetyl glutamate
NASDA
™
nucleic acid sequence-based
amplification
NHGRI
National Human Genome Research
Institute
NO
nitric oxide
PBSCT
peripheral blood stem-cell
transplantation
PCR
polymerase chain reaction
RCR
repair chain reaction
REF
Restriction endonuclease finger-
printing
SCN5A
a cardiac voltage-dependent sodium
channel &agr;-subunit gene
sec
second
SSCP
single strand conformation
polymorphism
SDA
strand displacement activation
WT
wild type
BACKGROUND ART
Cardiac arrhythmias are a cause of substantial morbidity and mortality in adults. A variety of therapeutic agents commonly used to treat arrhythmias, along with other non-cardiac drugs, sometimes provoke potentially dangerous disturbances of cardiac rhythm. Prolongation of the cardiac action potential by blocking the rapidly activating cardiac delayed rectifier potassium current, I
Kr
, is the desired therapeutic effect of many anti-arrhythmic agents, but is an inadvertent adverse effect of certain antihistamines, antidepressants, gastric motility promoters, and other agents. Generally without warning, 1-10% of patients receiving action potential prolonging drugs will develop marked prolongation of the electrocardiographic QT interval or polymorphic ventricular tachycardia (torsades de pointes). See Roden, D. M.,
N.Engl.J.Med.
331:785-791 (1994); Roden, D. M.,
Am J Cardiol
72:44B-49B (1993); Carlsson, L., et al.,
J. Pharmacol. Exp. Ther.
282:220-227 (1997); Mohammad, S., et al.,
Am. J. Physiol. Heart Circ. Physiol.
273:H2534-H2538(1997); Rampe, D., et al.,
FEBS Lett
417:28-32 (1997); Woosley, R. L., et al.,
JAMA
269:1532-1536 (1993); Suessbrich, H., et al.,
FEBS Lett
385:77-80 (1996); Weissenburger, J., et al.,
Clin Exp Allergy
29 (Suppl. 3):190-196 (1999); Jackman, W. M., et al.,
Prog Cardiovasc Dis
31:115-172 (1988); Lazzara, R.
Eur Heart J
14:H88-H92(1993); Tan, H. L., et al.,
Ann.Intern.Med.
122:701-714 (1995).
Congenital long QT syndrome (LQTS) is an inherited condition of abnormal cardiac repolarization characterized clinically by an increased risk of torsades de pointes. See Keating, M. T.
Medicine (Baltimore)
75:1-5 (1996); Vincent, G. M.
Annu. Rev. Med.
49:263-274 (1998); Roden, D. M., et al.,
Circulation
94:1996-2012 (1996). The majority of LQTS subjects appear to harbor mutations in either of two cardiac potassium channel genes, HERG and KCNQ1 (see Curran, M. E., et al.,
Cell
80:795-803 (1995) and Wang, Q., et al.,
Nature Genet
12:17-23 (1996)), while additional cases are caused by mutations in genes encoding potassium channel regulatory subunits KCNE1 and KCNE2 (see Splawski, I., et al.,
Nature Genet.
17:338-340 (1997); Abbott, G. W., et al.,
Cell
97:175-187 (1999)), a cardiac voltage-dependent sodium channel &agr;-subunit SCN5A (see Wang, Q., et al.,
Cell
80:805-811 (1995)), and other unidentified gene products (see Schott, J. J., et al., 57:1114-1122 (1995)).
A few anecdotal reports have attempted to associate the presence of rare sequence variants in KCNQ1 and KCNE2 to drug-induced LQT in the absence of an overt congenital phenotype. See Abbott, G. W., et al.,
Cell
97:175-187 (1999); Schulze-Bahr, E., et al.,
Circulation
96:1-211(1997); Napolitano, C., et al.,
Circulation
96:1-211(1997); Donger, C., et al.,
Circulation
96:2778-2781 (1997); Priori, S. G., et al.,
Eur Heart J
18:324(1997). However, in these cases, the described alleles were absent in the general population, indicating that they are only rare causes of drug-induced arrhythmia susceptibility. Thus, the prevalence of drug induced cardiac arrhythmias in the general population remains unexplained and uncharacterized.
Therefore, drug-induced cardiac arrhythmias continue to represent the Achilles heel of efforts to develop safe and effective anti-arrhythmic agents. Moreover, drug induced cardiac arrhythmias also occur during treatment with non-cardiac drugs that have unintended effects on cardiac repolarization. For example, as many as 10% of patients treated with quinidine, sotalol, and ibutilide will develop excessive QT interval prolongation or exhibit the precipitous occurrence of torsade de pointes. This unpredictable adverse reaction can occur in the absence of identifiable risks factors such as hypokalemia, hypomagnesemia, concomitant treatment with other I
Kr
blockers, and recent conversion from atrial fibrillation. See Tan, H. L., et al.,
Ann. Intern. Med.
122:701-714 (1995).
A method that can predict individual susceptibility to drug-induced arrhythmias would have substantial clinical utility and would meet a long-felt need in the art. However, such a method is currently not available in the art.
DISCLOSURE OF THE INVENTION
A method of screening for susceptibility to a drug-induced cardiac arrhythmia in a subject is disclosed. The method comprises: (a) obtaining a biological sample from the subject; and (b) detecting a D85N polymorphism of a KCNE1 gene encoding a cardiac potassium channel minK subunit polypeptide in the biological sample from the subject, the presence of the D85N polymorphism indicating that the susceptibility of the subject to a drug-induced cardiac arrhythmia.
Preferably, the polymorphism of the minK polypeptide comprises a G to A transition in the single exon of the KCNE1 gene, more preferably at nucleotide 281 of a cDNA that corresponds to the KCNE1 gene. More preferably, the G to A transition at nucleotide 281 of the cDNA that corresponds to the KCNE1 gene further comprises a change in the triplet code from GAC to AAC or GAT to AAT, which encodes a KCNE1 polypeptide having an asparagine (Asn or N) moiety at amino acid 85, instead of an aspartate (Asp or D) moiety. Hence, the polymorphism is referred to as the KCNE1-D85N polymorphism.
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 minK polypeptides and KCNE1 polynucleotides are also disclosed herein.
It is therefore an object of the present invention to provide polynucleotide molecules that can be used in analyzing a cardiac potassium channel minK subunit gene (KCNE1) in vertebrate subjects.
It is also an object of the present invention to provide for the determination of KCNE1 genotype in vertebrate subjects and particularly human subjects, based on information obtained through the analysis of nucleic acids, including genomic DNA and cDNA, derived from tissues from the subject.
It is yet another object of the present invention to provide a ready method for determining KCNE1 genotype.
It is still a further object of the present invention to provide polypeptide and polynucleotide molecules for use in generating antibodies that distinguish between the different fo
George, Jr. Alfred L.
Roden Dan M.
Jenkins & Wilson, P.A.
Myers Carla J.
Vanderbilt University
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