Described is a method for detecting 5-methylcytosine in genomic DNA samples. First, a genomic DNA from a DNA sample is chemically converted with a reagent, 5-methylcytosine and cytosine reacting differently, and the pretreated DNA is subsequently amplified using a polymerase and at least one primer. In the next step, the amplified genomic DNA is hybridized to at least two oligonucleotides, the latter being joined by inserting at least one oligonucleotide. In the ligation product, one nucleotide carries a detectable label, and the elongation depends on the methylation status of the specific cytosine in the genomic DNA sample. In the next step, the elongated oligonucleotides are analyzed for the presence of the label.

The present invention relates to a method for detecting 5-methylcytosine in genomic DNA-samples.
The levels of observation that have been well studied by the methodological developments of recent years in molecular biology are the genes themselves, the translation of these genes into RNA, and the resulting proteins. The question of which gene is switched on at which point in the course of the development of an individual, and how the activation and inhibition of specific genes in specific cells and tissues are controlled is correlatable to the degree and character of the methylation of the genes or of the genome. In this respect, the assumption suggests itself that pathogenic conditions express themselves in an altered methylation pattern of individual genes or of the genome.
The present invention describes a method for detecting the methylation state of genomic DNA samples. The method can, at the same time, also be used for detecting point mutations and single nucleotide polymorphisms (SNPs).
5-methylcytosine is the most frequent covalently modified base in the DNA of eukaryotic cells. It plays a role, for example, in the regulation of transcription, in genetic imprinting, and in tumorgenesis. Therefore, the identification of 5-methylcytosine as a component of genetic information is of considerable interest. However, 5-methylcytosine positions cannot be identified by sequencing since 5-methylcytosine has the same base pairing behavior as cytosine. Moreover, the epigenetic information carried by the 5-methylcytosines is completely lost during PCR amplification.
A relatively new, and currently the most frequently used method for analyzing DNA for 5-methylcytosine is based on the specific reaction of bisulfite with cytosine which, upon subsequent alkaline hydrolysis, is converted into uracil which corresponds to thymidine in its base pairing behavior. However, 5-methylcytosine remains unmodified under these conditions. Consequently, the original DNA is converted in such a manner that methylcytosine, which originally could not be distinguished from cytosine by its hybridization behavior, can now be detected as the only remaining cytosine using "normal" molecular biological techniques, for example, by amplification and hybridization or sequencing. All of these techniques are based on base pairing which can now be fully exploited. In terms of sensitivity the prior art is defined by a method which encloses the DNA to be analyzed in an agarose matrix, thus preventing the diffusion and renaturation of the DNA (bisulfite only reacts with single-stranded DNA), and which replaces all precipitation and purification steps with fast dialysis (Olek, A. et al, Nucl. Acids. Res. 1996, 24, 5064-5066). Using this method, it is possible to analyze individual cells, which illustrates the potential of the method. Until now, however, only individual regions of a length of up to approximately 3000 base pairs are analyzed; a global analysis of cells for thousands of possible methylation analyses is not possible. Moreover, this method cannot reliably analyze very small fragments from small sample quantities either. These are lost in spite of the diffusion protection by the matrix.
An overview of further methods of detecting 5-methylcytosines can be gathered from the following survey article: Rein, T., DePamphilis, M. L., Zorbas, H., Nucleic Acids Res. 1998, 26, 2255.,
With few exceptions (e.g., Zeschnigk M. et al, Eur. J. Hum. Genet. 1997, 5, 94-98), the bisulfite technology is currently only used in research. Always, however, short specific fragments of a known gene are amplified subsequent to a bisulfite treatment and either completely sequenced (Olek, A. and Walter, J., Nat. Genet. 1997, 17, 275-276) or individual cytosine positions are detected by a primer extension reaction (Gonzalgo, M. L., and Jones, P. A., Nucl. Acids Res. 1997, 25, 2529-2531, WO Patent 9500669) or by enzymatic digestion (Xiong, Z. and Laird, P. W., Nucl. Acids. Res. 1997, 25, 2532-2534). In addition, detection by hybridization has also been described (Olek et al., WO 99 28498).
Further publications dealing with the use of the bisulfite technique for methylation detection in individual genes are: Xiong, Z. and Laird, P. W. (1997), Nucl. Acids Res. 25, 2532; Gonzalgo, M. L. and Jones, P. A. (1997), Nucl. Acids Res. 25, 2529; Grigg, S. and Clark, S. (1994), Bioassays 16, 431; Zeschnik, M. et al. (1997), Human Molecular Genetics 6, 387; Teil, R. et al. (1994), Nucl. Acids Res. 22, 695; Martin, V. et al. (1995), Gene 157, 261; WO 97 46705, WO 95 15373 and WO 45560.
An overview of the Prior Art in oligomer array manufacturing can be gathered from a special edition of Nature Genetics (Nature Genetics Supplement, Volume 21, January 1999), published in January 1999, and from the literature cited there.
There are different methods known for immobilizing DNA. The best-known method is the fixed binding of a DNA which has been functionalized with biotin to a streptavidin-coated surface (Uhlen, M. et al. 1988, Nucleic Acids Res. 16, 3025-3038). The binding strength of this system corresponds to that of a covalent chemical bond without being one. To be able to covalently bind a target DNA to a chemically prepared surface, a corresponding functionality of the target DNA is required. DNA itself does not possess any functionalization which is suitable. There are different variants of introducing a suitable functionalization into a target DNA: two functionalizations which are easy to handle are primary aliphatic amines and thiols. Such amines are quantitatively converted with N-hydroxysuccinimide esters, and thiols react quantitatively with alkyl iodides under suitable conditions. The difficulty exists in introducing such a functionalization into a DNA. The simplest variant is the introduction via a PCR primer in a PCR. Disclosed variants use 5′-modified primers (NH2 and SH) and a bifunctional linker.
An essential component of the immobilization on a surface is its constitution. Systems described heretofore are mainly composed of silicon or metal. A further method of binding a target DNA is based on the use of a short recognition sequence (e.g., 20 bases) in the target DNA for hybridization to a surface-immobilized oligonucleotide. Enzymatic variants for introducing chemically activated positions in a target DNA have been described as well. In this case, a 5′-NH2-functionalization is carried out enzymatically on a target DNA.
For scanning an immobilized DNA array, fluorescently labeled probes have often been used. Particularly suitable for fluorescence labeling is the simple attachment of Cy3 and Cy5 dyes to the 5′-OH of the specific probe. The detection of the fluorescence of the hybridized probes is carried out, for example via a confocal microscope. Cy3 and Cy5 dyes, besides many others, are commercially available.
An overview of the Prior Art in oligomer array manufacturing can be gathered from a special edition of Nature Genetics (Nature Genetics Supplement, Volume 21, January 1999), published in January 1999, and from the literature cited there, as well as from U.S. Pat. No. 5,994,065 on methods for preparing solid supports for target molecules such a oligonucleotides at reduced, non-specific background signal.
More recent methods for detecting mutations are specified in the following:
Worth mentioning as a special case of sequencing is the single-base primer extension (Genetic Bit Analysis) (Head, S R., Rogers, Y H., Parikh K., Lan, G., Anderson, S., Goelet, P., Boycejacino M T., Nucleic Acids Research. 25(24): 5065-5071, 1997; Picoult-Newberg, L., Genome Res. 9(2): 167-174, 1999). A combined amplification and sequencing is described in U.S. Pat. No 5,928,906 where a base-specific termination on matrix molecules is used. A further method uses a ligase/polymerase reaction for identifying nucleotides (U.S. Pat. No. 5,952,174).
Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI) is a very efficient development for the analysis of biomolecules (Karas, M. and Hillenkamp, F. (1988), Laser desorption ionization of proteins with molecular masses exceeding 10000 daltons. Anal. Chem. 60: 2299-2301). An analyte is embedded in a light-absorbing matrix. Using a short laser pulse, the matrix is evaporated, thus transporting the analyte molecule into the vapor phase in an unfragmented manner. The analyte is ionized by collisions with matrix molecules. An applied voltage accelerates the ions into a field-free flight tube. Due to their different masses, the ions are accelerated at different rates. Smaller ions reach the detector sooner than larger ones.
MALDI is ideally suited to the analysis of peptides and proteins. The analysis of nucleic acids is somewhat more difficult (Gut, I. G. and Beck, S. (1995), DNA and Matrix Assisted Laser Desorption Ionization Mass Spectrometry. Molecular Biology: Current Innovations and Future Trends 1: 147-157.). The sensitivity for nucleic acids is approximately 100 times worse than for peptides and decreases disproportionally with increasing fragment size. For nucleic acids having a multiply negatively charged backbone, the ionization process via the matrix is considerably less efficient. For MALDI, the selection of the matrix plays an eminently important role. For the desorption of peptides, several very efficient matrixes have been found which produce a very fine crystallization. For DNA, there are currently several responsive matrixes in use, however, this has not reduced the difference in sensitivity. The difference in sensitivity can be reduced by chemically modifying the DNA in such a manner that it becomes more similar to a peptide. Phosphorothioate nucleic acids in which the usual phosphates of the backbone are substituted by thiophosphates can be converted into a charge-neutral DNA using simple alkylation chemistry (Gut, I. G. and Beck, S. (1995), A procedure for selective DNA alkylation and detection by mass spectrometry. Nucleic Acids Res. 23: 1367-1373). The coupling of a charge tag to this modified DNA results in an increase in sensitivity to the same amount as that found for peptides. A further advantage of charge tagging is the increased stability of the analysis against impurities which make the detection of unmodified substrates considerably more difficult.
Genomic DNA is obtained from DNA of cell, tissue or other test samples using standard methods. This standard methodology is found in references such as Fritsch and Maniatis eds., Molecular Cloning: A Laboratory Manual, 1989.
Mutualities between promoters consist not only in the occurrence of TATA- or GC-boxes but also for which transcription factors they possess binding sites and at what distance these are located from each other. The existing binding sites for a specific protein do not match completely in their sequence but conserved sequences of at least 4 bases are found which can still be elongated by inserting wobbles, i.e., positions at which in each case different bases are located. Moreover, these binding sites are present at specific distances from each other.
However, the distribution of the DNA in the interphase chromatin which occupies the largest portion of the nuclear volume is subject to a very special arrangement. Thus, the DNA is attached to the nuclear matrix, a filamentous pattern at the inner side of the nuclear membrane, at several locations. These regions are designated as matrix attachment regions (MAR) or scaffold attachment regions (SAR). The attachment has an essential influence on the transcription or the replication. These MAR fragments have no conserved sequences, but to 70% they consist of A or T, and are located in the vicinity of cisacting regions, which regulate the transcription in a general manner, and in the vicinity of topoisomerase II recognition sites.
In addition to promoters and enhancers, further regulatory elements, so-called "insulators", exist for different genes. These insulators can, for example, inhibit the action of the enhancer on the promotor if they are located between enhancer and promotor, or else, if located between heterochromatin and a gene, can protect the active gene from the influence of the heterochromatin. Examples of such insulators include: firstly, so-called "LCR" (locus control regions) consisting of several sites which are hypersensitive to DNAase I; secondly, certain sequences such as SCS (specialized chromatin structures) or SCS′, 350 or 200 bp long, respectively, and highly resistant to degradation by DNAase I, and flanked on both sides with hypersensitive sites (distance in each case 100 bp). The protein BEAF-32 binds to scs′. These insulators can be located on both sides of the gene.

Ligase/polymerase method for detecting cytosine methylation in DNA samples


A multilayer construction that includes a first layer that includes water sensitive thermoplastic polymer and a second layer disposed on the first layer, the second layer including superabsorbent polymer.

The invention is directed to a superabsorbent multilayer construction.
Superabsorbent polymers, which are available as powders, particles, and aqueous compositions, absorb large quantities of water and are often used in absorbent articles to increase the absorbency of the article. Disposable diapers and feminine hygiene products often include superabsorbent polymers to enhance body fluid absorption. Superabsorbent polymers also have various applications in the medical, food, and agricultural industries.

Superabsorbent water sensitive multilayer construction


A vehicle lamp includes a light source, and a projection lens which is configured to project light emitted from the light source. The projection lens includes two or more resin lenses and one or more glass lenses, and a refractive power ratio R (=Pr/Pt) of a total refractive power Pr of the resin lenses to a refractive power Pt of the entire projection lens satisfies a relationship of R<⅓.

As a headlamp of an automobile, there has been proposed an ADB light distribution control as one method for obtaining a light distribution for preventing dazzling to a vehicle (hereinafter referred to as a "front vehicle") in a front region of an own vehicle, such as a preceding vehicle or an oncoming vehicle in the front region, while increasing an illumination effect of the front region of the own vehicle. The ADB light distribution control includes detecting a front vehicle by a vehicle position detection device, reducing or turning off a light amount in a region in which the detected front vehicle presents, while brightly illuminating other wide regions.
In recent years, the ADB light distribution control is also applied to a headlamp using a light emitting element such as an LED as a light source. Specifically, in the headlamp, light from a plurality of LEDs as light sources, that is, illumination region of respective LEDs are combined to form a light distribution for illuminating the front region of the own vehicle. Further, when a front vehicle is detected, LEDs in an illumination region corresponding to the detected front vehicle are dimmed or turned off.
In the ADB light distribution control, white light emitted from the plurality of LEDs is projected to the front region of the own vehicle by a projection lens to form a plurality of illumination regions, these illumination regions are combined and synthesized appropriately, and thus an appropriate illumination region is formed. However, a pattern shape of the light of the LEDs to be projected may vary due to aberration caused by the projection lens, which makes it difficult to perform the ADB light distribution control with high accuracy.
In JP-A-2017-16928, a rear main surface of the projection lens is designed to have a predetermined curvature, so that a direction of coma aberration is specified and uniformity of the light pattern to be projected is improved. However, since the technique of JP-A-2017-16928 does not cope with the aberration, this technique would not cope with change in pattern shape of the light caused by the aberration.
In order to cope with the aberration, it is considered to configure the projection lens with a plurality of lenses, for example, a triplet lens. In this case, in order to reduce the weight and cost of the projection lens, it is also considered to configure a part of the plurality of lenses with resin lenses. For example, JP-A-H8-68935 proposes, a technique in which in a camera including a triplet lens, a first lens and a second lens are formed of resin and a third lens is formed of glass.
Since the lens of JP-A-H8-68935 is applied to a camera which is often used at a so-called normal temperature (or room temperature), a problem caused by change in ambient temperature would rarely arise. However, a problem may arise when this type of lens is applied to a projection lens of a lamp of an automobile. That is, when applied to a projection lens of a lamp of an automobile, since an ambient temperature varies in a range of 0° C. to 80° C. while the lamp is turned on and turned off, change of optical characteristics of the triplet lens due to change of thermal expansion of the lens formed of resin, in particular a spot shape due to spherical aberration is noticeable. When the spot shape formed by the projection lens changes with the temperature change, the pattern shape of the illumination region to be projected also changes, and therefore, the reliability of the ADB light distribution control may deteriorate with the temperature change.


The invention relates to a genetically modified infectious measles virus derived from a live-attenuated measles virus strain, in which the gene encoding the viral accessory C protein has been knocked out (MV-deltaC). It concerns in particular the use of said genetically modified infectious MV-deltaC in the treatment of malignant tumour or cancer conditions, and for the preparation of agents or compositions for such treatment.

Malignant mesothelioma is a rare and highly aggressive cancer, resistant to usual curative treatments. The development of the malignant pleural mesothelioma is mostly linked to a prolonged exposure to asbestos fibres and dusts (Kazan-Allen et al., Lung cancer, 2005, 49S1:S3-S8; Robinson et al., Lancet, 2005, 366:397-408). Melanoma is a malignant tumour that develops in melanocytes and can spread to the entire body when it is not treated. Although it represents one of the least frequent types of skin cancers, it is responsible for the most skin cancer-related deaths (Chin, L. et al., Genes Dev. 2006 20: 2149-2182). Lung adenocarcinomas are the most common kind of lung cancer, both in smokers and non-smokers and are one of the most common causes of cancer death (Travis, W. D. et al., J Thorac Oncol., 2011, 6(2), 244-285).
No strategy currently proposes a significant curative opportunity for several aggressive cancers such as malignant mesotheliomas, melanomas and lung adenocarcinomas.
Their prognoses are very poor and they are relatively refractory to all conventional treatment modalities, such as chemotherapy, radiotherapy and/or surgery. There is thus a pressing need for the development of new clinical approaches.
Cancer virotherapy is widely regarded as a new alternative for the treatment of cancers that are resistant to conventional anti-cancer therapies (Boisgerault N et al., Immunotherapy, 2010, march 2(2), 185-199). Oncolytic virotherapy has demonstrated multimodal antitumour mechanisms in both pre-clinical and a few phase I clinical anti-cancer treatments (Lech, P J and Russell, S J; Expert Review of Vaccines, 2010, 9(11):1275-1302; Galanis et al., Cancer Research, 2010, 70(3):875-882), and also in in vitro investigations (Gauvrit, A et al., Cancer Research, 2008, 68 (12), 4882-4892). Compared with cell-transfer immunotherapy, virus-vaccines have the advantage of conferring personalised anti-cancer immunity simultaneously with cytoreduction without the requirement for personalised manufacture. Additionally, virus-vaccines can be engineered to delete immunosuppressive viral components and to insert transgenes that enhance antitumour cytotoxicity and immunity (A. Gauvrit et al., Cancer Research, 2008, 68 (12), 4882-4892).
Measles virus (MV) is a non-segmented single-stranded, negative-sense enveloped RNA virus of the genus Morbilivirus within the family of Paramyxoviridae. The non-segmented genome of MV has an antimessage polarity, which results in a genomic RNA which is neither translated in vivo or in vitro nor infectious when purified. This virus has been isolated in 1954 (Enders, J. F. and Peebles, T. C., 1954, Proc Soc Exp Biol Med, 86(2): 277-286), and live-attenuated vaccines have been derived from this virus since then to provide vaccine strains, and in particular from the Schwarz/Moraten strain.
Transcription and replication of non-segmented (−) strand RNA viruses and their assembly as virus particles have been studied and reported especially in Fields virology (3rd edition, vol 1, 1996, Lippincott-Raven publishers—Fields B N et al.). Transcription and replication of MV do not involve the nucleus of the infected cells but rather take place in the cytoplasm of said infected cells. The genome of MV comprises genes encoding six major structural proteins from the six genes (designated N, P, M, F, H and L) and the additional two-non structural proteins from the P gene, the C and V proteins. The gene order is the following: 3′, N, P (including C and V), M, F, H, and L large polymerase protein at the 5′ end (FIG. 1A). The genome further comprises non-coding regions in the intergenic region M/F; this non-coding region contains approximately 1000 nucleotides of untranslated RNA. The cited genes respectively encode the leader peptide (I gene), the proteins of the nucleocapsid of the virus, i.e., the nucleoprotein (N), the phosphoprotein (P), and the large protein (L), which assemble around the genome RNA to provide the nucleocapsid. The other genes encode the proteins of viral envelope including the hemagglutinin (H), the fusion (F) and the matrix (M) proteins. The MV C protein is coded by the polycistronic P gene, it is a small (186 amino acids) and basic protein, localised both in the cytoplasm and in the nucleus (Bellini, W. J. et al., J. Vivol., 1985, 53:908-919). The role of this viral protein, described as a MV virulence factor, is not yet well understood. To determine the role of the MV C protein, a recombinant wild-type MV strain lacking expression of the C protein, based on the highly pathogenic IC-B strain and generated by using a reverse genetics system, has been used. It was suggested that the MV C protein could be involved in the assembly of viral particles, in the virus protein expression and in the delay of apoptosis of infected cells in order to establish a long term MV infection (Takeuchi, K. et al., J. Vivol., June 2005, 7838-7844). Although it has been reported that the MV C protein inhibits the interferon antiviral response (Shaffer, J. A. et al., Virology, 2003, 315:389-397), another study reached the opposite conclusion (Takeuchi, K. et al., J. Vivol., June 2005, 7838-7844), thereby confirming that the function of the MV C protein has not yet been well established.
Among human viruses that deserve to be tested as oncolytic agents, live-attenuated MV vaccine presents a number of advantages. Administered to hundreds of millions of children during 30 years, it is the safest and most widely used human paediatric vaccine. Attenuated strains of MV infect a large number of cell types and preferentially the transformed cancer cells. This is due to the use by MV of CD46 as a receptor frequently over-expressed in cancer cells to resist to the complement-dependant killing by natural killer cells (Naniche, D. et al., J Vivol, 1993, 67(10): 6025-6032; Dhiman, N. et al., Rev Med Vivol, 2004, 14(4): 217-229), whereas wild-type MV uses preferentially SLAM (CD150) (Tatsuo, H. et al. Nature, 2000, 406(6798):893-897; Anderson, B. D. et al., Cancer Res., 2004, 64: 4919-4926; Schneider, U. et al., J Virol., 2002, 76: 7460-7467). Interestingly, MV exhibits natural antitumour properties by specifically targeting cancer cells without infecting the healthy ones. Thus, MV demonstrates an unquestionable safety profile for application in future therapeutic protocols.
The oncolytic properties of wild-type MV are well known to a person skilled in the art (Mayo Foundation for Medical Education and Research, U.S. Ser. No. 07/854,928). Recently, clinical trials were initiated to investigate the capacity of an Edmonston MV strain to treat ovarian, glioblastoma, non-small lung cancer and multiple myeloma (see http://clinicaltrials.gov, measles and cancer keywords). The use of MV vaccines, either recombinant or chimeric, as vaccination vectors has also been described (WO2004/000876, WO2004/076619, WO2006/136697, and WO2008/078198).
This technology has also been proposed to the immuno-oncolytic treatment of mesothelioma (Gauvrit, A. et al., Cancer Research, 2008, 68 (12), 4882-4892). Accordingly, International Patent Application WO2009/047331 described both the oncolytic and immuno-adjuvant properties of the live-attenuated Schwarz strain of MV vaccine on a panel of epithelioid mesothelioma tumour cells. Using a rescued Schwarz strain of MV vaccine produced from an infectious cDNA clone, it was shown that MV-infected mesothelioma cells induced spontaneous monocyte-derived dendritic cell (Mo-DC) maturation and a tumour antigen-specific response.
The potential advantages of oncovirotherapy over conventional treatments include the property to induce an immune response including not only higher on-target specificity against cancer antigens (tumour associated antigens), and thus a better safety margin but also prolonged effect due to immune memory, and thus preventing relapse and metastasis. Indeed, it has been demonstrated that an immune-specific response and memory are developed after administration of MV at the site of cancer cells, in the presence of antigen-presenting cells (Massé, D. et al., Int. J. Cancer, 2004, 111(4), 575-580); Liu et al., Molecular therapy: the journal of the American Society of Gene Therapy, 2010, 18(6):1155-1162). It was demonstrated that the antitumour activity of the Schwarz MV strain acts through multiple mechanisms, including oncolysis, induction of tumour immunogenic apoptosis (danger signal expression associated with cell death) and virus-mediated syncytium formation (Gauvrit, A. et al., Cancer Res, 2008, 68(12), 4882-4892). In addition, the released tumour associated antigens and the inflammation resulting from viral replication have also been suggested to break the immunotolerance to tumours and induce anticancer immunity.
Despite an efficient infection, some MV-infected malignant tumour or cancer cells resist to cell death induction. Hence there is a need for the development of viruses that would help to overcome this type of resistance, and thus improve and extend the specific cell death induction of malignant tumour or cancer cells.
Dendritic cell (DC) precursors are divided into monocyte-derived dendritic cells (Mo-DCs) and plasmacytoid dendritic cells (pDCs), which display different functional properties. pDCs are a subset of DCs involved in the antiviral immune response due to their expression of Toll-like receptors (TLR) specialised in the recognition of viral nucleic acids (TLR7, TLR9) (Gilliet, M. et al., Nat Rev Immunol., 2008, 8:594-606). They respond to a wide range of viruses (inter alia influenza A virus, herpes simplex virus, HIV) in terms of activation and maturation by producing large amounts of type-I interferon (IFN-α,-β,-ω). They are also able to present viral antigens to CD8+ and CD4+ T cells when they are infected by a virus (Fonteneau, J. F. et al., Blood, 2003, 101:3520-3526) and to cross-present viral antigens from virus-infected cells to CD8+ T lymphocytes (Di Pucchio, T. et al., Nat Immunol., 2008, 9:551-557; Lui, G. et al., PLoS One, 2009, 4:e7111). It has also been shown that pDCs could play a beneficial role in the immune response against tumours (Drobits, B. et al., J Clin Invest., 2012, 122:575-585; Liu, C. et al., J Clin Invest., 2008, 118:1165-1175). As an example, in a mouse melanoma model, pDC activation and antitumour immune response were observed inside tumours after topical treatment with the TLR7 ligand, imiquimod (Drobits, B. et al., J Clin Invest., 2012, 122:575-585). As MV is single-stranded RNA (ssRNA), the inventors have hypothesised that pDCs could be able to detect the MV infection of tumour cells, because of their intravacuolar TLR7 expression which recognises single-stranded RNA.

Use of a genetically modified infectious measles virus with enhanced pro-apoptotic properties (MV-deltaC virus) in cancer therapy

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS)

INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM)


Compositions and methods based on a polymorphism in the gene encoding the xcex16 subunit of the human GABAA 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 xcex16 polypeptide sequence. Significantly, the polymorphism was associated with decreased sensitivity to ethanol and benzodiazepine drugs.

The present invention relates generally to the field of molecular genetics. More particularly, the invention relates to a polynucleotide sequence encoding a variant xcex16 subunit of the human GABAA neurotransmitter receptor, the sequence variant predicting sensitivity to both benzodiazepine drugs and ethanol.
Human heritability studies using twins and adoptees have indicated that alcoholism is a complex disorder having a genetic component. (Hesselbrock, xe2x80x9cThe Genetic Epidemiology of Alcoholismxe2x80x9d 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)).
xcex3-Aminobutyric acid (GABA) is a key inhibitory neurotransmitter in the mammalian CNS. GABA subtype A (GABAA) 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 GABAA receptor channels are heterooligomeric structures composed of several distinct polypeptide subunits (xcex11-6, xcex21-3, xcex31-3, and xcex4). (Burt et al., FASEB J 5:2916 (1991)).
Two lines of evidence have implicated the GABAA receptor in differential sensitivity to alcohol. First, cerebellar membranes of xe2x80x9cANTxe2x80x9d and xe2x80x9cATxe2x80x9d 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 GABAA xcex16 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 GABAA xcex16 receptor. (Korpi et al., Nature 361:356 (1993)).
In a second line of evidence, the GABAA xcex32 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 GABAA xcex32L 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.
Compositions and methods based on a polymorphism in the gene encoding the xcex16 subunit of the human GABAA 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 GABAA xcex16 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.
Herein, we have discovered two genetic differences or xe2x80x9cpolymorphismsxe2x80x9d that occur in the gene sequence encoding the xcex16 subunit of the human GABAA neurotransmitter receptor. In the most prevalent form of the xcex16 subunit of the human GABAA 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 xcex16 receptor subunit protein or the xcex16 receptor subunit protein-encoding polynucleotide having a proline residue at amino acid position 385 is referred to throughout this disclosure as xe2x80x9cPro385xe2x80x9d.
In this invention, we have discovered a first polymorphism, called xe2x80x9cPro385Serxe2x80x9d or xe2x80x9cSer385xe2x80x9d, in the xcex16 subunit of the human GABAA neurotransmitter receptor. This form of the xcex16 subunit of the human GABAA 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 xe2x80x9cPro385Serxe2x80x9d or xe2x80x9cSer385xe2x80x9d refers to a polymorphism in a polynucleotide encoding xcex16 protein (in which case the polymorphism is with reference to codon 385 of the xcex16-encoding polynucleotide), or to the xcex16 protein itself (in which case the polymorphism is with reference to amino acid position 385 of the xcex16 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 xcex16 protein (in which case the polymorphism is with reference to codon 385 of the xcex16 fragment-encoding polynucleotide), or to a fragment of the xcex16 protein itself (in which case the polymorphism is with reference to amino acid position 385 of the xcex16 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 greater than 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 xcex16 receptor. The G1031C polymorphism can also be referred to as GABRA6 1031G greater than C.
The Pro385Ser polymorphism occurs within a portion of the xcex16 subunit of the human GABAA neurotransmitter receptor corresponding to the second intracellular domain of the receptor near a putative protein kinase C phosphorylation site. By the phrase, xe2x80x9ca portion of the xcex16 subunit of the human GABAA neurotransmitter receptorxe2x80x9d is meant a segment of the xcex16 polypeptide sequence that includes at least 50-100 amino acids, more preferably at least 20 contiguous amino acids, and even more preferably at least 3, 6 or 10 contiguous amino acids. The Pro385Ser polymorphism exhibits a rarer-allele frequency of 0.08, whereas, the G1031C polymorphism exhibits a rarer-allele frequency of 0.47. By xe2x80x9cAllelexe2x80x9d or xe2x80x9callelic variantxe2x80x9d is meant the natural polynucleotide sequences corresponding to polymorphisms present in human beings.
In the following disclosure, we first 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 contrast, the G1031C polymorphism did not correlate with diazepam sensitivity. Second, 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.
Biological tools, therapeutics, and methods of use of the foregoing are provided. Further, embodiments that employ diagnostics or diagnostic kits, which are particularly useful for the rapid identification of the Pro385Ser polymorphism and, thus the determination of the relative sensitivity to a BZD drug or ethanol in an individual having a particular genotype, are provided. In the section below, we discuss the discovery of the Pro385Ser polymorphism in greater detail.
Genetic Polymorphisms in the GABAA Receptor xcex16 Subunit
Those having ordinary skill in the art will appreciate that differential sensitivity to benzodiazepine drugs is a useful indicator of brain differences correlating with behavioral variation, including susceptibility to alcoholism. In rats, for example, variation in alcohol and benzodiazepine sensitivity has been correlated with an inherited variant of the GABAA xcex16 receptor. Significantly, responses to eye movement tasks which can be measured as saccade gain, peak saccade velocity, and smooth pursuit gain, have been demonstrated to be reliable and reproducible within and between testing sessions, and to be objective, quantifiable measures affected by BZD in a dose-dependent manner. (Roy-Byrne et al., Psychopharmacology 110:85-91 (1993); Roy-Byrne et al., Biol Psychiatry 38:92 (1995)). Accordingly, in the procedures described below, eye movement testing provided a convenient means of monitoring BZD and ethanol sensitivity in human subjects.
Based on the finding that SOAs exhibited decreased sensitivity to BZD, and the fact that rodent genetic studies implicated GABAA receptors in response to BZD and alcoholism, we first investigated whether polymorphisms in the GABAA xcex16 subunit gene were associated with differences in BZD sensitivity in children of alcoholics. The procedures employed during the development of the invention involved correlating BZD sensitivity and genotype. More particularly, the procedures involved assessing sensitivity to diazepam in COAs using two eye movement tasks: peak saccadic velocity and average smooth pursuit gain. Genetic variation within the GABAA xcex16 receptor gene coding region was evaluated in 56 unrelated COAs by the single strand conformational polymorphism method. Association analysis using a Ser385 substitution, the synonymous variant G1031C and GABAA xcex16 haplotype was performed with smooth pursuit gain and peak saccadic velocity as dependent variables. As indicated below, the Ser385 genotype correlated with less diazepam-induced impairment in average smooth pursuit gain (t=1.954, df=53, p=0.04), but not peak saccadic eye velocity. The synonomous G1031C polymorphism was not associated with altered diazepam effects on either eye movement task.
Subjects in the studies described herein were 26 unrelated sons and 30 daughters of alcoholic fathers. All subjects were Caucasian. Subjects ranged from 18-25 years of age. Psychiatric disorders were diagnosed by DSM-III-R criteria and using the Structured Clinical Interview for DSM-III-R (SCID). (Spitzer et al., Structured Clinical Interview for DSM-III-R Patients Edition (SCID-P, 9/1/89 version), New York, Biometrics Research Department New York Psychiatric Institute, (1989)). The proband and at least one first-degree relative were interviewed with the Family Informant Schedule and Criteria. (Mannuzza et al., Family Informant Schedule and Criteria (FISC) Anxiety Disorders Clinic, New York, New York Psychiatric Institute, 1985), an extended version of the Family History-Research Diagnostic Criteria (Andreasen et al., Arch Gen Psychiatry 43:421 (1986)) and with the Family History Assessment Module from the SSAGA, a structured diagnostic interview used in the Collaborative Study on the Genetics of Alcoholism. (Bucholz et al., J Stud Alcohol 55:149 (1994)).
The modalities described above enabled us to confirm the diagnosis of alcohol dependence in the father, how many other first- and second-degree relatives were affected, and to establish family history of other psychiatric disorders. COAs were free of lifetime DSM-III-R Axis I or Axis II psychiatric or substance use disorders, except adjustment disorder and had no history of antisocial personality disorder. All COAs were medically healthy and by their report had taken no medication for at least one month nor had they used a benzodiazepine more than once. All subjects had negative urine drug screens, normal xcex3-glutamyltransferase, and normal mean corpuscular volume. Example 1 describes the methods that were used to measure BZD sensitivity in human subjects in greater detail. In the section below, we describe the association of diazepam sensitivity with the Pro385 and Ser385 polymorphisms.
Diazepam Sensitivity and the Pro385 or Ser385 GABAA xcex16 Genotype
Initially, the saccadic eye movement velocity and smooth pursuit eye movement gain was measured in the 56 COAs tested. Two baseline measurements for each variable were taken and averaged. The differences from this baseline were then evaluated at set time points after each of four diazepam doses. The data points were used to determine, by a trapezoidal technique, the integrated area under the resulting curve. The area under the curve for the dependent variable was then divided by the area under the curve for plasma diazepam levels to correct for individual variability in diazepam levels. The ratios of the areas under the curves for COAs with the Ser385 homozygous genotype and the Pro/Ser heterozygous genotype were then compared by t-test. There were no Ser/Ser homozygotes in the test population of COAs. For the G1031C genotype, a one-way ANOVA was used for assessing association.
The genotypes at each locus were also tested for Hardy-Weinberg equilibrium by Fisher""s Exact Test. A maximum likelihood method was used to estimate haplotype frequencies in the double heterozygotes. (Hill, Heredity 33:229 (1974); Weir, Genetic data analysis II, Sunderland, Mass., Sinauer Associates (1996)). Haplotype frequencies from single heterozygotes were determined by direct counting. Normalized linkage disequilibrium (xcex94) and linkage disequilibrium (D) were calculated using these haplotype frequencies.
The polymorphic variation in the GABAA xcex16 coding region from the 56 unrelated COAs was then confirmed using SSCP followed by DNA sequencing. (See Example 2). The two relatively abundant polymorphisms that were identified in the xcex16 coding region are presented in Table 4 (Table 4 can be found in Example 2, infra). A thymidine to cytosine transition at nucleotide 1236 of the coding sequence was identified and the transition results in a substitution of a proline residue by a serine residue at codon 385. This polymorphism was designated xe2x80x9cPro385Serxe2x80x9d or xe2x80x9cGABRA6 1236C greater than T. The second polymorphism identified was a silent G to C substitution at nucleotide 1031, and was designated xe2x80x9cG1031Cxe2x80x9d or GABRA6 1031G greater than C. As presented in the appended sequence listing, the Pro385 sequence is given by SEQ ID NO: 11, and the Ser385 sequence is given by SEQ ID NO: 12. The G1031C GABAA xcex16 sequence is given by SEQ ID NO: 13 while the C1031C sequence is given by SEQ ID NO: 14.
PCR-RFLP assays were also used to facilitate rapid genotyping based on the two GABAA xcex16 polymorphisms. (See Example 2). All subjects genotyped by SSCP-analysis and PCR-RFLP yielded identical results. The frequencies of the rarer Pro385Ser and 1031C alleles in the COAs were 0.08 and 0.47, respectively. Both genotype distributions were consistent with expectations based on Hardy-Weinberg equilibrium estimates. The level of linkage disequilibrium between Pro385Ser and G1031C was significant (xcex94=1.00, D=0.0401, "khgr"2=10.8, d.f.=1, p less than 0.0005).
An association between Ser385 genotype and average smooth pursuit eye movement gain following intravenous diazepam administration was observed (t=1.95, df=53, P=0.04). (See Table 1). COAs having the Pro385Ser allele exhibited a reduced sensitivity to diazepam relative to a group consisting of COAs having the Pro385 genotype. By reduced sensitivityxe2x80x9d is meant that a level of sensitivity that is lower than the level of sensitivity which characterizes individuals having the Pro385 genotype. The Pro385Ser allele, however, was not associated with the peak velocity of saccadic eye movement (t=0.171, df=52, p=0.865). Further, no association between the synonymous G1031C polymorphism and either of the two eye movement tasks was found. The numbers of subjects used in the two analyses differed because one individual was so sensitive to diazepam that data for saccadic velocity could not be collected at the highest diazepam dose.
Next, we looked to correlate polymorphic variants of the GABAA xcex16 receptor in COAs, specifically the Ser385 genotype, with saccadic eye movement velocity and smooth pursuit eye movement gain differences in COAs before and after diazepam administration, as disclosed in the section below.
The Pro385Ser Polymorphism and Benzodiazepine/Ethanol Insensitivity
Among the 13 subunits comprising the GABAA/BZD receptor complexes in mammals the xcex16 subunit is unique in its benzodiazepine agonist-insensitive pharmacology and in its restricted distribution. (Burt et al., FASEB J 5:2916 (1991)). GABAA xcex16 expression, for example, is limited to cerebellar granule cells. (Luddens et al., Neuropharmacology 34:245 (1995); Luddens et al., Nature 346:648 (1990)). In the genotyping procedure discussed above, we found that the level of response to alcohol in humans correlated with the GABAA xcex16 genotype. This discovery is the first indication that a difference in human diazepam response, and, thus, ethanol response can arise from a naturally occurring variant in a GABAA receptor subunit. Not wanting to be limited to any particular mechanism and offered only for the purposes of explanantion, we believe that diazepam binding to the xcex16 Ser385 receptor modulates GABAA currents, which results in the differences in smooth pursuit eye movement gain observed between subjects having the Pro385 genotype.
We did not observe an association between the Ser385 genotype and saccadic eye movement. Saccadic eye movements involve the parietal cortex, the frontal cortex and associated connections with the basal ganglia, as well as the superior colliculus and, finally, pre-motor areas in pons. (Henn et al., Rev Neurol 145:540 (1989)). Pursuit eye movements that were associated with Pro385Ser are predominantly mediated by multiple cortical areas and cerebellum. (Leigh et al., The Neurology of Eye Movement. 2 ed. Philadelphia, F. A. Davis Co., 1991). Hence, saccadic velocity and smooth pursuit gain provide a measure of BZD effects in brainstem and cortex/cerebellum, respectively. It is noteworthy that the association of Pro385Ser to pursuit eye movements but not to saccadic movements corresponds to the restricted expression of GABAA xcex16 to the cerebellum.
We also did not find any association between G1031C and diazepam sensitivity in spite of the fact that Pro385Ser and G1031C are in strong linkage disequilibrium. Although linkage disequilibrium between the two loci is strong, allele frequency differences between the two loci can account for lack of association with the more abundant G1031C synonymous variant.
Pro385 is conserved in the GABAA xcex16 receptors of rats and mice (Luddens et al., Nature 346:648 (1990)) (Kato, J Mol Biol 214:619 (1990)). The Ser385 amino acid residue is located in the second intracellular domain of the receptor and at a site ten amino acid residues removed from a consensus phosphorylation site for PKC. (Luddens et al., Nature 346:648 (1990)). Some believe that the domain can contribute to subtype specificity and intracellular regulatory mechanisms. (Olsen et al., FASEB J 4:1469 (1990)). Until the present disclosure, however, a function for this amino acid and domain has not been confirmed.
With respect to the GABAA xcex32L subunit, it is of interest that an alternatively spliced variant of xcex32 which contains an extra eight amino acids bears a consensus site for phosphorylation by PKC. (Whiting et al., Proc Natl Acad Sci USA 87:9966 (1990)). Site-directed mutagenesis of the alternatively spliced sequence indicates that the xcex32L subunit must be phosphorylated to confer ethanol sensitivity. (Wafford et al., Neuron 7:27 (1991); Wafford et al., FEBS Lett 313:113 (1992)). Not wanting to be limited to any particular mechanism and offered only for the purposes of explanation, we speculate that a similar role for this domain of the xcex16 subunit could enable the Pro385Ser variant to alter the sensitivity of the receptor to ethanol and BZD.
After determining that individuals with the Pro385Ser polymorphism exhibited a reduced sensitivity to benzodiazepine drugs when compared with patients having the Pro385 polymorphism, we verified that individuals with the Pro385Ser polymorphism also exhibited a reduced sensitivity to ethanol when compared with patients having the wild-type polymorphism. These results are described in the following section.
Individuals with the Pro385Ser Polymorphism Exhibit a Reduced Sensitivity to Ethanol
Alcohol abuse and dependence (alcoholism) are complex disorders that appear to reflect several genetic influences that together might explain 40% to 60% of the variance of risk. (Kendler et al., Arch Gen Psychiatry 54:178-184 (1997); Pickins et al., Arch Gen Psychiatry 48:19-28 (1991)). A low level of response (LR) to alcohol appears to be one route by which vulnerability is mediated. Even after controlling for other influences, LR is apparently both genetically influenced and associated with an enhanced risk for alcohol dependence. Schuckit and Smith, Arch Gen Psychiatry 53:202-210 (1996)). In humans, LR has been found to occur at a higher frequency among young drinking, but nonalcoholic, family history positive (FHP) men (Pollock Am J Psychiatry 149:1534-1538 (1991); Schuckit and Smith 1996; Newlin and Thomson Psychol Bull 108:383-402 (1990)), while identical twins are more similar on LR than fraternal twins. (Madden et al 1995; Rose et al 1994). Three follow-ups of subjects from alcohol challenges have revealed higher rates of alcoholism, alcohol-intake, or problems for individuals with low LR. (Rodriguez et al., Alcohol Clin Exp Res 17:155-161 (1993); Schuckit and Smith (1996); Volovka et al Arch Gen Psychiatry 53:258-263 (1996)). In addition, our group has reported a correlation for LR of 0.3 across two generations of subjects. Animal studies support the contention that genetic influences impact on LR (Crabbe et al. J Pharmacol Exp Ther 277:624-632 (1996)), and in some studies, rodents with low LR have been noted to consume higher amounts of alcohol. (Gill et al., Alcohol Clin Exp Res 21:106A (1997); Li et al., Behavior Genetics 23:163-170 (1993)).
As part of a larger study, 41 men, about 39 years old, were selected from among the first 113 completed 15-year follow-ups in a prospective study. The genotyping was performed selectively from the first group of consecutively ascertained sons of alcoholics and controls from among what will eventually be a larger population as part of an ongoing study. Seventeen subjects whose low level of response to ethanol (LR) at age 20 were in the lower third were compared on five polymorphisms of four genes with 24 men whose reactions to alcohol had been above the median. Fourteen men with the LL genotype of the serotonin transporter (5-HTT) polymorphism and seven men with the Ser385 genotype of the GABAA (xcex16) polymorphism were found to have demonstrated lower LR scores at about age 20 and were significantly at greater risk of alcoholism than the other genotypes for those loci. All four subjects with combined LL and Ser385 genotypes had developed alcoholism and demonstrated the lowest LR scores overall. There was no evidence that two polymorphisms of the 5-HT2A receptor gene and one of the 5-HT2C receptor gene were related to LR or alcoholism in this sample.
For this pilot study, blood samples were taken from the first 41 appropriate consecutive men among the first 113 who completed 15-year follow-ups in an ongoing study. In order to identify subjects who were clearly low on LR, the men were selected to represent the two poles of LR. These included the first 17 subjects who upon original alcohol challenge at about age 20 demonstrated an overall LR in the lowest third, and the 24 whose intensities of reaction to alcohol had been above the median (High LR). The alcohol challenges, evaluated changes from baseline for subjective feeling of intoxication, standing steadiness, and hormones after drinking 0.75 ml/kg of ethanol. (Schuckit and Gold, Arch Gen Psychiatry 45:211-216 (1986). The overall LR was evaluated as the change from baseline (pre-alcohol) to the time of peak blood alcohol concentration (BAC) (60 minute values) following alcohol consumption.
Previously, about ten years after initial testing, all 453 original subjects were located, and 450 (99.3%) were successfully evaluated (Schuckit and Smith 1996). The current pilot data were gathered as part of the ongoing 15-year follow-up of the same sample. For genotyping, blood was drawn at the time of the 15-year interview, and analyses performed blind to the phenotypic classification of the subjects. Five polymorphisms were genotyped from genomic DNA prepared from lymphoblastoid cell lines as follows. For 5HT2a T102C and His452Tyr, using methods described elsewhere, polymorphisms were typed by PCR and restriction enzyme assays (Ozaki et al., Biol Psychiatry 40:1267-1272 (1996)). 5HT2c Cys23Ser was genotyped by creating an artificial restriction site using a PCR primer that introduces a base substitution close to the codon of interest (Haliassos et al., Nucleic Acids Res. 17:3606 (1989)), and for 5HTTLPR, DNA amplification was accomplished using the two flanking primers used by Heils et al. (J. Neurochem 66:2621-2624 (1996)). This set of primers amplifies a 484 or 528 bp fragment corresponding to the 5HTTLPR short or long allele. The Ser385 polymorphism was genotyped using primers GABAaxcex16-9f and GABAaxcex16-9b: 5xe2x80x2CTG ACT CCA AAT ATC ATC TG3xe2x80x2 (SEQ. ID. NO. 9) and 5xe2x80x2GAG AAG CAT CTA CAC AAG TC3xe2x80x2 (SEQ. ID. NO. 10). Amplification with this set of primers resulted in a 367 bp PCR product containing a Fok I restriction site.
Genotypes for each of the five polymorphisms were evaluated against the three dependent variables provided in Table 2, with diagnoses based on the criteria of the Third Revised Diagnostic and Statistical Manual (DSM-III-R) of the American Psychiatric Association (1987). The differences across the two or three genotypes available at each locus were evaluated either by ANOVA or Student""s t-test for continuous variables, and chi-square (X2) with Yates"" correction for categorical data.
The 41 males selected for low and high LR were similar in age (about 39 years), marital status (about 73% married), and education (only 5% lacked a college education). Consistent with prior reports (Schuckit and Smith 1996), the low LR subjects were significantly more likely to have been diagnosed as alcohol dependent during the 15-year follow-up (64.7% vs. 8.3%, "khgr"2=14.6, df=1, p less than 0.002). There were no significant differences in the self-reported ethnic group of origin ("khgr"2=7.16, df=5, p=0.21), and all subjects were Caucasian.
Table 2 presents the genotype by trait analyses for each candidate gene locus. For the 5-HTT polymorphism, significantly lower LR. scores were observed at age 20 for the 14 subjects with the LL genotype (p=0.04). Consistent with these observations, subjects with the LL genotype were significantly more likely to have fulfilled criteria for alcohol abuse or dependence at some time during the 15 years of follow-up (p=0.04), but there were no differences across groups in the proportion with alcoholic relatives. Because of evidence that the 5-HTT S allele might act in a dominant fashion (Heils et al J Neurochem 66:2621-2624 (1996)), the combined SL and SS genotype groups were compared to the LL group, with results supporting an enhanced and statistically significant difference on the mean LR (xe2x88x920.89xc2x11.00 vs 0.16xc2x10.75, t=2.64, df=39, p=0.01), and on the proportion with an alcoholic diagnosis (57.1% vs 18.5%, X2=6.35, df=1, p=0.02). While not shown in the Table due to overlap with the mean LR score, we have also evaluated the proportion of subjects whose LR fell into the lowest third for comparability to prior publications. This proportion was also significantly higher for the LL group (71.4% LL, 31.6% SL, and 12.5% SS; X2=8.7, df=2, p=0.02.
Associations of the GABAAxcex16 genotype to both LR and alcoholism were also observed. Here, Pro/Ser heterozygous individuals were more likely than Pro385 homozygotes to be alcoholic (p=0.02), and they also demonstrated fronds for a lower LR score (p=0.06) and a higher proportion of FHPs (p=0.07). While not shown in Table 2, those with Ser385 were more likely to be in the lowest third in response to alcohol (71.4% vs. 35.3%, X2=3.12, df=1, p=0.08). Comparisons across the genotypes for the 5-HTT102C, 5-HT2Atyr His452tyr, and the 5-HT2c Cys23Ser polymorphisms demonstrated no relationship of genotypes to either LR scores or alcoholism diagnoses.
An analysis was also carried out combining information regarding the 5-HTT and GABAAxcex16 polymorphisms. The four men with a combination of both LL 5-HTT and Ser385 GABAA xcex16 genotypes (the two associated most closely with low LR and alcoholism) had the lowest LR at age 20 (xe2x88x921.29xc2x10.53 vs. xe2x88x920.74xc2x11.13 for LL/PP, 0.59xc2x11.11 for SL/PS, xe2x88x920.13xc2x10.88 for SL/PP and 0.03xc2x10.45 for SS/PPxe2x80x94test for linear trend t=2.86, df=4, p less than 0.007). All four of these subjects fell into the lowest third of LR, all were alcoholic, and all were FHP. At the other extreme, the eight men carrying both the SS 5-HTT and the Pro385 GABAA receptor genotypes (the two genotypes associated with the highest LR scores and nonalcoholic status) had the highest overall LR score, and were the least likely to be alcoholic. There was no evidence of an interaction between the 5-HTT and GABAAxcex16 genotypes (F(1,36)=0.01, p=0.92). As was true in the single locus analysis, these two-locus results reach even higher levels of statistical significance if all subjects with at least one copy of the dominant S allele are combined into one group.
The analysis above demonstrates the significant relationship to LR and alcoholism at two loci, the 5-HTTLPR serotonin transporter variant and the GABAA xcex16 amino acid substitution Ser385. Evidence supporting the importance of the GABAA receptor system in the effects of alcohol and the development of alcoholism is scant. The results above verify that the Pro385Ser polymorphism is indicative of LR and future alcoholism. Furthermore, the disclosure above clearly indicates that genotyping of the human xcex16 subunit of the GABAA receptor gene is useful for predicting whether an individual will exhibit a particular sensitivity to ethanol and/or benzodiazepine drugs.
The Existence of More Polymorphisms Associated with BZD and Ethanol Insensitivity
Those having ordinary skill in the art will appreciate that direct gene analysis using the SSCP technique may not be sufficiently sensitive to detect all possible sequence variants of the GABAA xcex16 receptor gene. In view of the fact that 40% of the GABAA xcex16 receptor gene coding sequence was not screened in this study, it is possible that unknown variants in either the coding sequence or promoter region of the xcex16 gene are in linkage disequilibrium with the Pro385Ser and G1031C variants described herein. In light of the findings presented above and the disclosure which follows, the discovery of more GABAA xcex16 receptor polymorphisms that are associated with ethanol and Benzodiazepine drug sensitivty would be routine. Regardless of the particular polymorphism used as a marker of genetically determined sensitivity to ethanol and benzodiazepine drugs, our findings indicate that methods based on xcex16 genotyping in humans can determine a predisposition to benzodiazepine/ethanol sensitivity. The section below describes many approaches that can be used to identify more polymorphisms in the GABAA xcex16 receptor gene that lead to BZD and ethanol insensitivity.
Characterization of Pro385Ser by Computational Analysis
Preliminary computational analysis revealed that the Pro385Ser polymorphism occurs in the second intracellular domain of the receptor near a putative protein kinase C phosphorylation site. Homology searches of nucleic acid and protein databases using software known to those of skill in the art can reveal other transmembrane proteins with a similar motif and can provide a greater understanding of how the Pro385Ser polymorphism confers BZD/ethanol insensitivity. Furthermore, by employing conventional approaches in protein modeling and methods of rational drug design, models of the three-dimensional structure of Pro385 or Ser385 GABAA xcex16 can be obtained and agents that interact or circumvent Pro385 or Ser385 GABAA xcex16 can be identified. Examples 3-6 disclose several software and hardware embodiments and provide computational methods that can be used to further characterize the Pro385 or Ser385 GABAA xcex16 nucleic acid and polypeptide sequences and develop drugs that interact with the Pro385 or Ser385 GABAA xcex16 receptors.
Aspects of the invention also include recombinant vectors, probes, and primers comprising Pro385 or Ser385 GABAA xcex16 sequence. The discussion below describes embodiments having Pro385 or Ser385 GABAA xcex16 nucleic acids.
Use of Nucleic Acids Encoding Pro385 or Ser385 GABAA xcex16, or Portions Thereof
The sequence of the Pro385 and Ser385 GABAA xcex16 is provided in the sequence listing (SEQ. ID NOs. 11 and 12). Wild-type and/or mutant Pro385 or Ser385 GABAA xcex16 sequences, their functional equivalents, or fragments of these sequences at least six nucleotides in length encoding Pro385 or Ser385 GABAA xcex16 can be used in accordance with embodiments of the invention. Preferably, the nucleic acid embodiments comprise at least 12, 15, or 17 consecutive nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom). More preferably, the nucleic acid embodiments comprise at least 20-30 consecutive nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom). In some cases, the nucleic acid embodiments comprise more than 30 nucleotides from the nucleic acids encoding Pro385 or Ser385 GABAA xcex16, or portions thereof. In other cases, the nucleic acid embodiments comprise at least 40, at least 50, at least 75, at least 100, at least 150, or at least 200 consecutive nucleotides from the nucleic acids encoding Pro385 or Ser385 GABAA xcex16, or portions thereof. The nucleic acids listed in SEQ. ID Nos. 9-10 are desirable embodiments, for example. Subsequences comprising hybridizable portions of Pro385 or Ser385 GABAA xcex16 sequence have use, e.g., in nucleotide acid hybridization assays, Southern and Northern Blot analysis, etc., as will be described infra.
Some embodiments comprise recombinant nucleic acids having all or part of the Pro385 or Ser385 GABAA xcex16 gene. A recombinant construct can be capable of replicating autonomously in a host cell. Alternatively, the recombinant construct can become integrated into the chromosomal DNA of the host cell. Such a recombinant polynucleotide comprises a polynucleotide of genomic or cDNA, of semi-synthetic or synthetic origin by virtue of human manipulation. Therefore, recombinant nucleic acids comprising sequences otherwise not naturally occurring are provided by embodiments of this invention. Although Pro385 or Ser385 GABAA xcex16 as it appears in nature can be employed, it will often be altered, e.g., by deletion, substitution, or insertion and will be accompanied by sequence not present in a human.
The nucleotide acid sequence depicted in the sequence listing (SEQ. ID NO. 12) can be altered by mutation such as substitutions, additions, or deletions that provide for sequences encoding functionally equivalent molecules. According to one embodiment, a molecule is functionally equivalent or active compared with a molecule having the sequence depicted in SEQ. ID NO: 11 or 12 if it has the ability to confer ethanol or BZD insensitivity. Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as derived from the sequence listing (SEQ. ID NO: 11 or 12) can be used in some embodiments of the present invention. These include, but are not limited to, nucleic acid sequences comprising all or portions of the Pro385 or Ser385 GABAA xcex16 gene which have been altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
In addition, recombinant Pro385 or Ser385 GABAA xcex16-encoding nucleic acid sequences of the invention can be engineered so as to modify processing or expression of Pro385 or Ser385 GABAA xcex16. For example, and not by way of limitation, the Pro385 or Ser385 GABAA xcex16 gene can be combined with a promoter sequence and/or ribosome binding site, or a signal sequence can be inserted upstream of Pro385 or Ser385 GABAA xcex16 encoding sequences to permit secretion of Pro385 or Ser385 GABAA xcex16 and thereby facilitate harvesting or bioavailability. Additionally, a given Pro385 or Ser385 GABAA xcex16 nucleic acid can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction sites or destroy preexisting ones, or to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson et al., J. Biol. Chem. 253:6551 (1978)).
By using the Pro385 or Ser385 GABAA xcex16 nucleic acid sequences disclosed in the sequence listing (SEQ ID NO. 12), probes can be designed and manufactured by oligonucleotide synthesis and cDNA or genomic libraries can be screened so as to isolate natural sources of the nucleic acid embodiments and homologs thereof. Alternatively, such nucleic acids can be provided by amplification of sequences resident in genomic DNA or other natural sources by PCR. Example 7 describes the preparation of PCR primers and the amplification of Pro385 or Ser385 GABAA xcex16 DNA.
The nucleic acids of the invention can also be used as reagents in isolation procedures and diagnostic assays. For example, sequences from nucleic acids encoding Pro385 or Ser385 GABAA xcex16, or portions thereof can be detectably labeled and used as probes to isolate other sequences capable of hybridizing to them. In addition, sequences from nucleic acids encoding Pro385 or Ser385 GABAA xcex16, or portions thereof can be used to make PCR primers by conventional oligonucleotide synthesis for use in isolation and diagnostic procedures. The discussion that follows describes some of the expression constructs and protein embodiments of the invention.
Pro385 or Ser385 GABAA xcex16 Peptides and Their Expression
Pro385 or Ser385 GABAA xcex16 proteins, or fragments, or derivatives thereof include, but are not limited to, those containing as a primary amino acid sequence all or part of the amino acid sequence substantially as deduced from the sequence listed in SEQ. ID NO: 11 or 12 including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. Accordingly, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs. For example, the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In other aspects of the invention, Pro385 or Ser385 GABAA xcex16 proteins or fragments or derivatives thereof, which are differentially modified during or after translation, e.g., by phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, linkage to an antibody molecule, membrane molecule, or other ligand, are contemplated. (Ferguson et al., Ann. Rev. Biochem. 57:285-320 (1988)).
In one embodiment, the inventors contemplate Pro385 or Ser385 GABAA xcex16 or a portion thereof, in a cell line. Further, the present inventors envision isolating or purifying Pro385 or Ser385 GABAA xcex16 protein. Example 8 provides several approaches to synthesize, express, and isolate or purify the Pro385 or Ser385 GABAA xcex16 protein, or fragments thereof. The term xe2x80x9cisolatedxe2x80x9d requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or protein present in a living cell is not isolated, but the same nucleic acid or protein, separated from some or all of the coexisting materials in the natural system, is isolated. In accordance with this definition, Pro385 or Ser385 GABAA xcex16 nucleic acid or protein or Pro385 or Ser385 GABAA xcex16 nucleic acid or polypeptide fragments present in a cell lysate are xe2x80x9cisolatedxe2x80x9d. The term xe2x80x9cpurifiedxe2x80x9d does not require absolute purity; rather it is intended as a relative definition. For example, recombinant nucleic acids and proteins are routinely purified to electrophoretic homogeneity, as detected by ethidum bromide staining or Coomassie staining, and are suitable in several assays despite having the presence of contaminants. Example 8 provides several approaches to synthesize, express, and isolate or purify the Pro385 or Ser385 GABAA xcex16 protein, or fragments thereof. Following synthesis or expression and purification of the proteins encoded by the Pro385 or Ser385 GABAA xcex16 nucleic acid or portion therof, the purified proteins can be used to generate antibodies as described in the following section.
Production of an Antibody to a Pro385 or Ser385 GABAA xcex16 Polypeptide
The antibodies contemplated have many uses including, but not limited to, biotechnological applications, therapeutic/prophylactic applications, and diagnostic applications. While antibodies capable of specifically recognizing the protein of interest can be generated using synthetic 15-mer peptides having a sequence encoded by Pro385 or Ser385 GABAA xcex16 gene or portion thereof by injecting the synthetic peptides into mice to generate antibody, a more diverse set of antibodies can be generated using recombinant or purified Pro385 or Ser385 GABAA xcex16 protein or fragments thereof, as described in Example 9. The discussion that follows describes several diagnostic embodiments of the invention.
Diagnostic Embodiments
Generally, the diagnostics and methods of use thereof can be classified according to whether the diagnostic detects the presence of Pro385 or Ser385 GABAA xcex16 nucleic acid in a sample or Pro385 or Ser385 GABAA xcex16 protein in a sample. Accordingly, the detection of the Pro385 or Ser385 GABAA xcex16 nucleic acid and/or protein in a biological sample indicates a predilection to BZD/ethanol insensitivity. Additionally, the manufacture of kits which incorporate the reagents and methods described in the following embodiments so as to allow for the rapid detection of the Pro385Ser polymorphism are contemplated. The diagnostic kits can include a nucleic acid probe or an antibody that specifically detects the Pro385Ser polymorphism, for example, or can detect both wild-type and mutant. The detection component will typically be supplied in combination with one or more of the following reagents. A substratum capable of absorbing or otherwise binding DNA, RNA, or protein will often be supplied. Available substrata for this purpose includes membranes of nitrocellulose, nylon or derivatized nylon that can be characterized by bearing an array of positively charged substituents. One or more restriction enzymes, such as FokI, can be furnished in the kit, as can non-human polynucleotides like calf-thymus or salmon-sperm DNA.
Useful nucleic acid-based diagnostic techniques include, but are not limited to, fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern Blot analysis, single-stranded confirmation analysis (SSCA), RNase protection assay, dot blot analysis, and PCR. The starting point for these analysis is isolated or purified DNA from a biological sample. Most simply, blood is drawn from the subject to be tested and DNA extracted from the nucleated cells in the blood. In some cases, primers corresponding to regions of Pro385Ser can be used with PCR to amplify this DNA so that it can be more easily be detected in diagnostic applications.
Several methods can be used to detect the Pro385Ser polymorphism in a biological sample. Direct DNA sequencing, either manual sequencing or automated fluorescent sequencing can detect sequence variation. Another approach is the single-stranded confirmation polymorphism assay (SSCA) (Orita et al., Proc. Natl. Acad. Sci. USA 86:2776-2770 (1989)) discussed above. This method, however, does not detect all sequence changes, especially if the DNA fragment size is greater than 200 base pairs, but can be optimized to detect most DNA sequence variation. The reduced detection sensitivity is a disadvantage, but the increased throughput possible with SSCA makes it an attractive, viable alternative to direct sequencing for mutation detection. The fragments which have shifted mobility on SSCA gels are then sequenced to determine the exact nature of the DNA sequence variation. Other approaches based on the detection of mismatches between the two complimentary DNA strands include clamped denaturing gel electrophoresis (CDGE) (Sheffield et al., Am. J. Hum. Genet. 49:699-706 (1991)), heteroduplex analysis (HA) (White et al., Genomics 12:301-306 (1992)), and chemical mismatch cleavage (CMC) (Grompe et al., Proc. Natl. Acad. Sci. USA 86:5855-5892 (1989)). A review of currently available methods of detecting DNA sequence variation can be found in a recent review by Grompe, Nature Genetics 5:111-117 (1993).
A rapid preliminary analysis to detect polymorphisms and DNA sequences can be performed by looking at a series of Southern Blots of DNA cut with one or more restriction enzymes preferably with a large number of restriction enzymes. Each block contains lanes of DNA from uninfected individuals and the DNA to be tested. Southern Blots displaying hybridizing fragments when probed with sequences corresponding to Pro385Ser indicate the presence of the genotype associated with BZD/ethanol insensitivity. Detection of point mutations can also be accomplished by amplifying the DNA directly from the sample using primers corresponding to the regions of Pro385Ser by standard PCR techniques. The DNA sequence of the amplified sequences can then be determined.
There are six well-known methods for confirming the presence of Pro385Ser: 1) single-stranded confirmation analysis (SSCA) (Orita et al.); 2) denaturing gradient gel electrophoresis (DGGE) (Wartell et al., Nucl. Acids Res. 18:2699-2705 (1990), Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236 (1989), 3) RNase protection assays (Finkelstein et al., Genomics 7:167-172 (1990), Kinszler et al., Science 251:1366-1370 (1991)); 4) the use of proteins which recognize nucleotide mismatches, such as the E. Coli mutS protein (Modrich, Ann. Rev. Genet. 25:229-253 (1991); and allele-specific PCR (Rano and Kidd, Nucl. Acids Res. 17:8392 (1989)). For allele-specific PCR, primers are used which hybridize at their 3xe2x80x2 ends to the Ser385 GABAA xcex16 mutation. If the particular Ser385 mutation is not present, an amplification product is not observed. Amplification Refractory Mutation System (ARMS) can also be used, as disclosed in European Patent Application Publication No. 0332435 and in Newton et al., Nucl. Acids Res. 17:2503-2516 (1989).
In the first methods (SSCA, DGGE, and RNase protection assay), a new electrophoretic band appears. SSCA detects a band which migrates differentially because the sequence change causes a difference in single-strand, intramolecular base pairing. RNase protection involves cleavage of the mutant polynucleotide into two or more smaller fragments. DGGE detects differences in migration rates of sequences compared to wild-type sequences, using a denaturing gradient gel. In an allele-specific oligonucleotide assay (ASOs) (Conner et al., Proc. Natl. Acad. Sci. USA 80:278-282 (1983)), oligonucleotide is designed which detects a specific sequence, and an assay is performed by detecting the presence or absence of hybridization signal. In the mutS assay, the protein binds only to sequences that contain a nucleotide mismatch in a heteroduplex between mutant and wild-type sequences.
Mismatches, according the present invention, are hybridized nucleic acid duplexes in which the two strands are not 100% complementary. Lack of total homology can be due to deletions, insertions, inversions, or substitutions. Mismatched detection can be used to detect point mutations in the gene or in its mRNA product. While these techniques are less sensitive than sequencing, they are simpler to perform on a large number of biological samples.
An example of a mismatch cleavage technique is the RNase protection method. In practice, the method involves the use of a labeled riboprobe which is complementary to the Ser385 gene coding sequence. The riboprobe and either mRNA or DNA isolated from the biological sample are annealed (hybridized) together and subsequently digested with the enzyme RNase A which is able to detect some mismatches in a duplex RNase structure. If a mismatch is detected by RNase A, it cleaves at the site of the mismatch. Thus, when the annealed RNA is separated on a electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNase A, an RNA product will be seen which is much smaller than the full length duplex RNA for the riboprobe and the mRNA or DNA. The riboprobe need not be the fall length of Pro385 or Ser385 GABAA xcex16 mRNA or gene, it will be desirable to use a number of these probes to screen the whole mRNA sequence for mismatches. In a similar fashion, DNA probes can be used to detect mismatches, through enzymatic or chemical cleavage. See, e.g., Cotton, et al., Proc. Natl. Acad. Sci. USA 85:4397 (1988), Shenk et al., Proc. Natl. Acad. Sci. USA 72:989 (1975), Novack et al., Proc. Natl. Acad. Sci. USA 83:586 (1986).
Alternatively, mismatches can be detected by shifts in the electrophoretic ability of mismatched duplexes relative to matched duplexes. (See, e.g., Cariello, Human Genetics 42:726 (1988)). With either riboprobes or DNA probes, the cellular mRNA or DNA which might contain the Ser385 gene can be amplified by PCR before hybridization. DNA sequences isolated from biological samples which have been amplified by use of PCR can be screened using allele-specific probes. These probes are nucleic acid oligomers, each of which contains a region of Ser385 sequence. For example, one oligomer can be about 30 nucleotides in length and corresponds to a portion of the Ser385 sequence referenced in SEQ ID NO. 12 By use of a battery of such allele-specific probes, PCR amplification products can be screened to identify the presence of the Pro385Ser polymorphism. Hybridization of allele-specific probes with amplified Pro385 or Ser385 GABAA xcex16 sequences can be performed, for example, on a nylon filter.
The most definitive test for the presence of the Pro385Ser polymorphism, is to directly compare nucleotide or protein sequences isolated from a biological sample with those from a control population. The control population, for example, can contain DNA, RNA, cDNA nucleic acids samples representative of the wild-type sequence, as listed in SEQ ID NO. 11 (experimental being the Pro385Ser polymorphism, as listed in SEQ ID NO. 12) Alternatively, one could sequence messenger RNA after amplification, e.g., by PCR. Examples 10-13 describe nucleic acid-based diagnostic methods of the invention.
In addition to the diagnostics and methods based on the detection of nucleic acid that encodes to Pro385 or Ser385 GABAA xcex16, diagnostics and methods based on the detection of Pro385 or Ser385 GABAA xcex16 proteins are also envisioned. The following discussion details several embodiments of this aspect of the invention.
Protein-Based Diagnostic Embodiments
The presence of Pro385 or Ser385 GABAA xcex16 protein can be detected by screening for the presence of the protein using conventional assays. For example, monoclonal antibodies immunoreactive with Pro385 GABAA xcex16 protein can be used to screen biological samples for the presence of the Pro385Ser polymorphism. Similarly, antibodies specific for or Ser385 GABAA xcex16 protein can be used to screen for the presence of the Pro385Ser polymorphism. Such immunological assays can be done in many convenient formats.
In a preferred embodiment, antibodies will immunoprecipitate specifically the Ser385 GABAA xcex16 protein (e.g., will not cross-react with the Pro385 protein) from solution as well as react specifically with Ser385 protein on Western or Immunoblots of polyacrylamide gel. In another preferred embodiment, antibodies will detect Ser385 in paraffin or frozen sections, using immunocytochemical techniques. Preferred embodiments relating to methods for detecting the Pro385 or Ser385 GABAA xcex16 protein include enzyme-linked immunosorbant assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies. Exemplary sandwich assays are described by David et al., in U.S. Pat. Nos. 4,376,110 and 4,486,530, hereby incorporated by reference.
We also contemplate the preparation of diagnostic kits comprising antibodies specific for the Ser385 protein and not cross-reactive with the Pro385 protein. Such kits can also include nitrocellulose or nylon membranes for immobilizing protein from a biological sample to be tested. Results from the kit assays can be interpreted by a healthcare provider or a diagnostic laboratory. Alternatively, diagnostic kits are manufactured and sold to private individuals for self-diagnosis. In addition, we contemplate the design and manufacture of supports having Pro385 or Ser385 GABAA xcex16 polypeptides, as described below, for use in methods for identifying agents that interact with the Pro385 or Ser385 GABAA xcex16 polypeptides.
Construction of a Multimeric Support Having Pro385 or Ser385 GABAA xcex16 or Polypeptide Fragments Thereof
A biotechnological tool that is useful for the discovery of agents that interact with Pro385 or Ser385 GABAA xcex16 or circumvent Pro385 or Ser385 GABAA xcex16 (e.g., high throughput screening) desirably provide Pro385 or Ser385 GABAA xcex16 in such a form or in such a way that a sufficient affinity for the agent is obtained. For example, while monomeric Pro385 or Ser385 GABAA xcex16 (i.e. appearing as a discrete unit of the protein on a support) is sufficiently active to mediate association with an agent, multimeric Pro385 or Ser385 GABAA xcex16 (i.e. appearing as multiple units of the protein on a support) can have a far greater affinity for the agent and would provide sufficient binding events to be detectable by conventional methods. Example 14 provides several approaches that can be used to create a multimeric support having Pro385 or Ser385 GABAA xcex16 or polypeptide fragments thereof. The multimeric supports can be used in methods of high throughput screening, several of which are described in the following section.
High Throughput Screening for Agents That Interact With Pro385 or Ser385 GABAA xcex16 Protein, or Polypeptide Fragments Thereof
High throughput screening is an approach to drug discovery that searches for a protein, peptide, peptidomimetic, or chemical that will interact on a defined target such as the Pro385 or Ser385 GABAA xcex16 protein, or a polypeptide fragment thereof. Generally, a library of proteins, polypeptides, peptides, peptidomimetics, or chemicals, collectively referred to as xe2x80x9cagentsxe2x80x9d, is screened against the target in biological assays and agents which interact with the target are identified and used directly as therapeutics or as a basis to develop new therapeutics using combinatorial chemistry and protein modeling. Example 15 provides several high throughput screening methods that are used to identify agents that interact with Pro385 or Ser385 GABAA xcex16 protein or polypeptide fragments thereof.
Example 1 describes the methods that were used to measure BZD sensitivity in human subjects.

Polynucleotides encoding polymorphic human GABAA receptor xcex1-6 subunit

The United States of America as represented by the Department of Health and Human Services


Methods, systems, and computer readable media are disclosed for simulating a circuit. The method may comprise a step of providing a network model of the circuit having a plurality of ports, the plurality of ports being associated with one or more net pairs. The method may also comprise combining the plurality of ports into one or more groups based on the net pairs, each group corresponding to a net pair. In addition, the method may comprise calculating, for each group, one or more expansion elements, wherein the one or more expansion elements are associated with a shared property among all ports of the group. Moreover, the method may comprise simulating the circuit using a combination of the expansion elements calculated for each group.

In electronic circuit design, simulations are often performed before physical prototypes are constructed. During simulations involving electronic package and/or multi-layer printed circuit board (PCB), physical systems are usually represented or approximated by mathematical models, and the mathematical models often contain high-order linear- and/or non-linear systems, which are complicated and difficult (or time-consuming) to solve. Accordingly, there is a need to effectively reduce the order of mathematical models to improve speed and efficiency.

Methods, systems, and computer-readable media for model order reduction in electromagnetic simulation and modeling


A cable connector configured to receive a sound signal and alter the tonality of the sound by switching among a plurality of capacitors. The cable connector comprises a switch coupled to a binary coded plurality of capacitors. The cable connector receives an input signal corresponding to a sound through a conductor, which is also coupled to the switch. The switch may be used to select one of a plurality of capacitances, affecting the tonality of the sound. A cable implementing the cable connector and a method for using the cable connector are also disclosed.

The present invention relates generally to an instrument interconnect cable, and more particularly to an instrument interconnect cable wherein the user can adjust the cable's capacitance.
Guitars and other stringed instruments are often amplified by attaching a passive magnetic pickup in close proximity to the vibrating metallic strings of the guitar and connecting this pickup to an amplifier and speaker using a cable. The vibrating strings change the magnetic flux within the core of the pickup. This flux change induces a voltage change in the outer coils of the pickup, thus completing the translation of mechanical vibration to electrical signal. This signal is transmitted through the cable to the amplifier.
The cable connecting the pickup to the amplifier is typically between 5 and 30 feet to allow the musician adequate mobility while playing. The cable typically is terminated on each end with a standard ¼′ male connector. Both the guitar and amplifier will typically have the mating female ¼″ connector.
Instrument cables are typically of the coaxial type, wherein the signal is surrounded by a 360-degree metallic wire shield. This type of cable protects the very low voltage signal coming from the instrument from radio frequency interference (RFI) or electro-magnetic fields (EMF).
Some musicians have noticed that different brands of instrument cables at different lengths will cause a difference in tonality in an instrument's sound output. One cause for this variance in tonality is the variance in the overall capacitance of the cables being used. Capacitance in cables is caused by the insulating material having a dielectric quality that causes capacitive coupling between the signal wires and the ground, or shield, wires. Coaxial cables are typically given a capacitance per foot rating (pF/ft). This rating typically can be anywhere from 10 pF/ft to 50 pF/ft. By multiplying the pF/ft rating by the length of the cable, a person can calculate the overall capacitance of a particular length of a particular cable. The overall capacitance of a cable typically ranges from 300 pF to 1200 pF. Cables of different capacitances will have different tonal qualities when used with an instrument having passive magnetic pickups.
With present cables, musicians are able to vary the capacitance only by using different cables of different lengths. This is inconvenient for several reasons. First, if a musician wants to produce the same tonality from his or her instrument, then he or she must use the same cable with the same length all the time, or the musician must try to find two or three cables of different lengths that have the same capacitance, exchanging one for the other whenever a cable of a different length is needed. Additionally, if a musician would like to take advantage of the different tonalities offered by cables having different capacitances, then he or she must undertake the inconvenient process of unplugging one cable and then plugging in another having a different capacitance to replace it. The current options available for addressing this issue do not offer a sufficient range of adjustment for altering the capacitance.
It can readily be appreciated that there is a need for an instrument cable that avoids the problems described above. The present invention fulfills this need and provides further related advantages.

Cable connector having high resolution adjustable capacitance and method for using the same


A stream reproducing device performing automatic viewing of a non-viewed period of a stream. The stream reproducing device comprises a camera a person detecting unit detecting the viewer based on the output of the camera, a viewing information generating unit generating viewing information based on the person detecting unit, and a reproduction control unit receiving supply of a stream, to control reproduction of the stream. The person detecting unit detects each of the viewers by classifying based on the output of the camera. The viewing information generating unit generates the viewing information for each of the viewers based on the detection result of each of the viewers. The viewing information generated by the viewing information generating unit is related to a time stamp of the stream to identify a non-viewed period, for which each viewer does not view the reproduced result of the stream.

Document 1 (Published Japanese patent application H08-18900) discloses a technology which detects whether a viewer is present or not in front of a stream reproducing device and performs automatic recording of the stream while the viewer is not present.
More specifically, a television receiver disclosed by Document 1 possesses the structure shown in FIG. 21. In FIG. 21, a terrestrial broadcasting receiving antenna 201 receives a signal of a terrestrial broadcasting. A satellite broadcasting receiving antenna 202 receives a signal of satellite broadcasting. A signal input terminal 203 receives an externally-fed audio and video signal.
A receiving unit 204 consists of a channel selection tuner and a demodulator circuit. The receiving unit 204 performs a channel selection of a specific station from the broadcast wave which antennas 201 and 202 receive, demodulates the selected signal, and generates an audio and video signal. A signal processing circuit unit 205 performs processing for the audio and video signal fed from the receiving unit 204 or the signal input terminal 203, and outputs the processed signal to a signal control unit 206.
A recording and reproducing device unit 207 possesses a recording medium and performs recording and reproduction of an audio and video signal. The reproduced audio and video signal is outputted to the signal control unit 206.
A detection device unit 208 detects whether any viewer is present or not in front of the television receiver, and outputs the detection result to the signal control unit 206.
A display unit 210 displays an image by the video signal. A speaker unit 209 converts a sound signal into sound. Parts other than the antennas 201 and 202 are housed in one case as the television receiver.
When the television receiver is turned on and brought to the state ready to be viewed, the detection device unit 208 detects whether there is any viewer present in front of the television receiver, and outputs the detection result to the signal control unit 206.
While the signal control unit 206 is inputting the detection result indicating the presence of a viewer from the detection device unit 208, the signal control unit 206 does not allow a recording and reproducing device unit 207 to operate. However, when the detection result indicates that the viewer is gone, the signal control unit 206 instructs the recording and reproducing device unit 207 to record the audio and video signal which the viewer has been viewing.
Afterward, when the viewer returns in front of the television receiver and the detection device unit 208 detects that the viewer is present again, the signal control unit 206 instructs the recording and reproducing device 207 to stop recording and to reproduce the recorded audio and video signal. Thereby, the viewer can view the stream for the non-viewed period automatically.
However, in the prior art, unless the audio and video signal itself during the non-viewed period is automatically recorded by the recording and reproducing device unit 207, the viewer cannot view the stream for the non-viewed period later. Therefore, when the remaining recordable time of the recording and reproducing device unit 207 is few or almost zero, the viewer cannot view the stream for the non-viewed period later.
In a place such as a living room (the television receiver is often installed at such a place), people often come and go. In the prior art, when at least one viewer is staying in front of the television receiver, automatic recording during the non-viewed period is not performed. Thus, when a plurality of viewers come and go alternately, and different non-viewed periods exist, the prior art can not handle this situation at all. After all, each of the viewers needs to view the stream for the non-viewed period, by manually finding his/her own non-viewed period and performing troublesome operation such as rewinding and fast-forwarding. Therefore, the prior art has practical difficulty in meeting the theme of automatic viewing of the stream for the non-viewed period later on.