Polynucleotides encoding polymorphic human GABAA receptor...

Details Polynucleotides encoding polymorphic human GABAA receptor... Polynucleotides encoding polymorphic human GABAA receptor...

2001-08-15

2004-07-13

Landsman, Robert (Department: 1647)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S006120

Reexamination Certificate

active

06762294

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to the field of molecular genetics. More particularly, the invention relates to a polynucleotide sequence encoding a variant &agr;6 subunit of the human GABA
A
neurotransmitter receptor, the sequence variant predicting sensitivity to both benzodiazepine drugs and ethanol.
BACKGROUND OF THE INVENTION
Human heritability studies using twins and adoptees have indicated that alcoholism is a complex disorder having a genetic component. (Hesselbrock, “The Genetic Epidemiology of Alcoholism” in
The Genetics of Alcoholism
, Edited by Begleiter H, Kissin B. New York, Oxford University Press, pp 17-39 (1995)). Sons of alcoholics (SOAs) are a group at high risk for developing alcoholism (Cloninger et al.,
Arch Gen Psychiatry
38:861 (1981)), and so have been the focus of numerous studies on the subjective, psychomotor, physiological and biochemical responses to ethanol. (Schuckit,
Alcohol Clin Exp Res
12:465 (1988); Newlin et al.,
Psychol Bull
108:383 (1990); Pollock,
Am J Psychiatry
149:1534 (1992)). These studies have identified several differences between SOAs and male control subjects which can provide clues to the basis of increased risk of developing alcoholism.
One of the distinctions between SOAs and male control subjects relates to differential sensitivity to benzodiazepine drugs (BZD) and ethanol. More particularly, SOAs have been shown to be significantly less sensitive to BZD (Ciraulo et al.,
Am J Psychiatry
146:1333 (1989); Cowley et al.,
Alcohol Clin Exp Res
16:1057 (1992); Cowley et al.,
Alcohol Clin Exp Res
18:324 (1994)) and ethanol (Schuckit et al.,
Arch Gen Psychiatry
53:202 (1996)) when compared with male control subjects. The genetic and neurobiological mechanisms underlying this diminished sensitivity is unclear, because these drugs affect multiple neurotransmitter systems in the central nervous system. (CNS) (Deitrich et al.,
Pharmacol Rev
41:489 (1989)).
&ggr;-Aminobutyric acid (GABA) is a key inhibitory neurotransmitter in the mammalian CNS. GABA subtype A (GABA
A
) receptors are chloride channels that specifically bind benzodiazepine drugs with high affinity (Luddens et al.,
Neuropharmacology
34:245 (1995)) to result in chloride ion influx. Molecular analysis has revealed that GABA
A
receptor channels are heterooligomeric structures composed of several distinct polypeptide subunits (&agr;1-6, &bgr;1-3, &ggr;1-3, and &dgr;). (Burt et al.,
FASEB J
5:2916 (1991)).
Two lines of evidence have implicated the GABA
A
receptor in differential sensitivity to alcohol. First, cerebellar membranes of “ANT” and “AT” rats exhibited differential affinity for BZD. (Uusi-Oukari et al.,
J Neurochem
54:1980 (1990)). The Alcohol-sensitive ANT (Alcohol Non-Tolerant) and alcohol-insensitive AT (Alcohol Tolerant) lines of rats have been selectively bred to exhibit differences in sensitivity to ethanol-induced motor impairment. An amino acid substitution Arg100Glu in the GABA
A
&agr;6 receptor is believed to be at least partially responsible for the difference in alcohol sensitivity which characterizes these two rat lines. The alcohol insensitive AT line carries the Arg100 form of the receptor and is diazepam insensitive when compared with the Glu100 GABA
A
&agr;6 receptor. (Korpi et al.,
Nature
361:356 (1993)).
In a second line of evidence, the GABA
A
&ggr;2 subunit was implicated in the differential sensitivity of long-sleep (LS) and short-sleep (SS) mouse lines to acutely administered alcohol. (Wafford et al.,
Science
249:291 (1990)). An in vitro mutagenesis and expression system employing Xenopus oocytes was used to demonstrate that alternative RNA splicing of a region of the GABA
A
&ggr;2L subunit, which encodes a consensus protein kinase C (PKC) phosphorylation site, was critical for modulation by ethanol. (Wafford et al.,
Neuron
7:27 (1991); Wafford et al.,
FEBS Lett
313:113 (1992)). Clearly there remains a need to better understand the genetic basis for differential sensitivity to benzodiazepine drugs and alcohol in humans.
SUMMARY OF THE INVENTION
Compositions and methods based on a polymorphism in the gene encoding the &agr;6 subunit of the human GABA
A
neurotransmitter receptor are disclosed. This polymorphism results in the substitution of a serine residue for a proline residue ordinarily present at amino acid position 385 of the GABA
A
&agr;6 polypeptide sequence. In a first set of experiments, we demonstrate that patients having the Pro385Ser polymorphism exhibit a reduced sensitivity to diazepam, a benzodiazepine drug known in the art to mimic a subject's response to ethanol, when compared with patients having the proline residue at amino acid position 385. The Pro385Ser polymorphism was found to be associated with less change in smooth pursuit eye movement gain after intravenous diazepam was administered to children of alcoholics (COAs). In a second set of experiments, we demonstrate that patients having the Pro385Ser polymorphism exhibit a reduced sensitivity to ethanol when compared with patients having the proline residue at amino acid position 385. Thus, the polymorphism was associated with decreased sensitivity to ethanol and benzodiazepine drugs.
DETAILED DESCRIPTION OF THE INVENTION
Herein, we have discovered two genetic differences or “polymorphisms” that occur in the gene sequence encoding the &agr;6 subunit of the human GABA
A
neurotransmitter receptor. In the most prevalent form of the &agr;6 subunit of the human GABA
A
neurotransmitter receptor, a proline residue is present at amino acid position 385 given by the sequence provided by Hadingham et al.,
Mol Pharmacol
49(2):253-259 (1996), herein incorporated by reference. This form of the &agr;6 receptor subunit protein or the &agr;6 receptor subunit protein-encoding polynucleotide having a proline residue at amino acid position 385 is referred to throughout this disclosure as “Pro385”.
In this invention, we have discovered a first polymorphism, called “Pro385Ser” or “Ser385”, in the &agr;6 subunit of the human GABA
A
neurotransmitter receptor. This form of the &agr;6 subunit of the human GABA
A
neurotransmitter receptor is characterized by a substitution of a serine residue for the proline residue which is ordinarily present at amino acid position 385. In some contexts, the term “Pro385Ser” or “Ser385” refers to a polymorphism in a polynucleotide encoding &agr;6 protein (in which case the polymorphism is with reference to codon 385 of the &agr;6-encoding polynucleotide), or to the &agr;6 protein itself (in which case the polymorphism is with reference to amino acid position 385 of the &agr;6 polypeptide sequence given by Hadingham et al.,
Mol Pharmacol
49(2):253-259 (1996). In other contexts, the term Pro385Ser or Ser385 refers to a polymorphism in a polynucleotide encoding a fragment of &agr;6 protein (in which case the polymorphism is with reference to codon 385 of the &agr;6 fragment-encoding polynucleotide), or to a fragment of the &agr;6 protein itself (in which case the polymorphism is with reference to amino acid position 385 of the &agr;6 polypeptide sequence given by Hadingham et al.,
Mol Pharmacol
49(2):253-259 (1996). The Pro385Ser polymorphism can also be referred to as GABRA6 1236C>T to indicate the single nucleotide change at position 1236 which confers the substitution of the serine amino acid residue for the proline amino acid residue at amino acid position 385.
We have also discovered a second polymorphism, called G1031C, but this nucleotide change did not alter the amino acid sequence of the &agr;6 receptor. The G1031C polymorphism can also be referred to as GABRA6 1031G>C.
The Pro385Ser polymorphism occurs within a portion of the &agr;6 subunit of the human GABA
A
neurotransmitter receptor corresponding to the second intracellular domain of the receptor near a putative protein kinase C phosphorylation site. By the phrase, “a portion of the &agr;6 subunit of the human GABA
A
neurotransmitter receptor” is meant a segment of the &agr;6 polypeptide sequence that includes

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