EP1183350A2 - Produits d'expression de genes impliques dans des affections du metabolisme du cholesterol - Google Patents

Produits d'expression de genes impliques dans des affections du metabolisme du cholesterol

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Publication number
EP1183350A2
EP1183350A2 EP00931354A EP00931354A EP1183350A2 EP 1183350 A2 EP1183350 A2 EP 1183350A2 EP 00931354 A EP00931354 A EP 00931354A EP 00931354 A EP00931354 A EP 00931354A EP 1183350 A2 EP1183350 A2 EP 1183350A2
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EP
European Patent Office
Prior art keywords
sequence
nucleic acid
seq
sequence seq
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP00931354A
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German (de)
English (en)
French (fr)
Inventor
Patrice Denefle
Marie-Françoise Rosier-Montus
Isabelle Arnould-Reguigne
Catherine Prades
Christian Clepet
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Aventis Pharma SA
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Aventis Pharma SA
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Publication date
Priority claimed from FR9906587A external-priority patent/FR2794131B1/fr
Application filed by Aventis Pharma SA filed Critical Aventis Pharma SA
Publication of EP1183350A2 publication Critical patent/EP1183350A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

  • the present invention relates to nucleic acids expressed from genes located in the human genome in region 9q31-34 of chromosome 9, which may be involved in diseases genetically linked to this chromosomal locus, in particular affections of the metabolism of plasma lipoproteins. , especially the reverse transport of cholesterol.
  • the invention also relates to polypeptides encoded by some of these nucleic acids as well as antibodies directed specifically against such polypeptides, useful as diagnostic reagents.
  • the invention relates to vectors and recombinant host cells comprising these nucleic acids or fragments thereof.
  • Lipoproteins, protein complexes incorporating lipids allowing the transport of these in the blood circulation, normally present in the blood plasma vary in size and composition but all appear in the form of microemulsion.
  • the lipoprotein particles are spherical and contain a central core of non-polar lipids (mainly triglycerides and cholesterol esters) and a surface monolayer of polar lipids (cholesterol and mainly phospholipids) and proteins called apolipoproteins (apo) .
  • apolipoproteins have amphipathic properties.
  • the association of polar lipids and proteins with lipoproteins is thus carried out via hydrophobic forces thanks to which the fatty acid chains and the side chains of non-polar amino acids are excluded from the aqueous environment.
  • Most apolipoproteins have helical amphipatic regions (apolipoprotein A1, A-II, A-IV, Cl, C-ll, 0-111 and E).
  • the density of the lipoprotein particles is inversely proportional to their size, their density reflecting the relative amounts of low density non-polar lipids contained in the nucleus and high density surface proteins present.
  • chylomicrons secreted by enterocytes, in which apo B-48 is predominant
  • VLDL secreted by hepatocytes, which contain the protein apo B-100.
  • LDL and HDL The smallest classes of lipoproteins, LDL and HDL, mainly contain cholesterol esters in their nucleus.
  • the mature forms of these particles are not secreted directly from the cells but are more particularly produced by metabolic pathways within the blood plasma.
  • LDL particles represent the end products of VLDL particle metabolism.
  • HDL particles Some components of HDL particles are derived from chylomicrons.
  • High density lipoproteins are one of the four major classes of lipoproteins that circulate in the blood plasma.
  • lipoproteins are involved in different metabolic pathways such as lipid transport, bile acid formation, steroidogenesis, cell proliferation and in addition interfere with plasma proteinase systems.
  • HDL are perfect acceptors of free cholesterol and, in combination with cholesterol ester transfer proteins (CETP), lipoprotein lipase (LPL), hepatic lipase (HL) and lecithin: cholesterol acyltransferase (LCAT), play a major role in the reverse transport of cholesterol, i.e. the transport of excess cholesterol in peripheral cells to the liver for its elimination from the body in the form of bile acid.
  • HDL not only transports cholesterol from peripheral cells to the liver, but also distributes it to steroid-producing cells or to cholesterol-depleted peripheral cells.
  • HDL precursors are secreted in discoid form from the intestine and liver, from which spherical particles are formed through the formation of cholesterol esters which migrate to the heart of the lipoprotein particle.
  • the nascent HDL particles contain apo A-l and apo A-IV, while the nascent hepatic HDL particles are rich in apo A-l, apo E and apo A-Il.
  • the lipid part of these particles consists of phospholipids and small amounts of free cholesterol and triglyceride. HDL has been shown to play a central role in the transport of cholesterol from peripheral tissues to the liver.
  • HDL high-density lipoprotein
  • HDL high-density lipoprotein
  • the protective role of HDL in reverse cholesterol transport is confirmed by epidemiological studies demonstrating an inverse relationship between cholesterol concentrations in these HDL and the risk of developing coronary heart disease, or by observations that HDL accept effectively excess intracellular cholesterol from various cell types.
  • Atherogenic lipoproteins are ingested by macrophages or peripheral cells and degraded in lysosomes. Cholesterol is released from lysosomes and is re-esterified in the cytoplasmic compartment.
  • HDLs rich in apo Al stimulate cholesterol flows from macrophages or peripheral cells to the extracellular compartment, following a interaction with HDL binding proteins present on the surface of these cells.
  • HDL deficiency Various diseases related to HDL deficiency have been described, including Tangier's disease, HDL deficiency and LCAT deficiency.
  • LPL lipoprotein lipase
  • apoC-11 activator apoC-11
  • RNA molecules were expressed from sequences localized in the genome in a region of approximately 15 cM centered on the microsatellite marker D9S1784, identified as the microsatellite marker giving the binding strongest genetics with family HDL deficiency and Tangier disease.
  • region 9q31-34 considered potentially contains genes capable of playing a role in the initiation or the development of various diseases, such as: - bone diseases such as myxoid chondrosarcomas, mental retardations linked to abnormalities of chromosome 9 (MRD);
  • NPH2 childhood nephrophtisia
  • LGMD2H muscular dystrophy of belts
  • schizophrenia - psychiatric illnesses
  • the messenger RNAs and the corresponding polypeptides are potentially implicated in some of the human pathologies described above, namely in other pathologies also genetically linked to this region of chromosome 9.
  • the present invention thus describes polynucleotides and polypeptides whose alteration of the sequence or expression is potentially associated with a deficit in the metabolism of plasma lipoproteins, more particularly with a deficit in the reverse transport of HDL.
  • the present invention also describes polynucleotides and polypeptides whose alteration in the sequence or expression is potentially associated with diseases genetically linked to locus 9q31-34 of chromosome 9.
  • isolated in the sense of the present invention designates a biological material (nucleic acid or protein) which has been removed from its original environment (the environment in which it is naturally located). For example, a polynucleotide naturally occurring in a plant or animal is not isolated. The same polynucleotide separated from adjacent nucleic acids within which it is naturally inserted into the genome of the plant or animal is considered to be “isolated”.
  • Such a polynucleotide may be included in a vector and / or such a polynucleotide may be included in a composition and nevertheless remain in an isolated state since the vector or the composition does not constitute its natural environment.
  • purified does not require that the material be present in a form of absolute purity, exclusive of the presence of other compounds. Rather, it is a relative definition.
  • a polynucleotide is in the "purified” state after purification of the starting material or of the natural material of at least one order of magnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.
  • the expression “nucleotide sequence” can be used to denote either polynucleotide or a nucleic acid.
  • the term “nucleotide sequence” encompasses the genetic material itself and is therefore not limited to information regarding its sequence.
  • nucleic acid include RNA, DNA, cDNA sequences or even RNA / DNA hybrid sequences of more than one nucleotide, in single chain form or in duplex form.
  • nucleotide denotes both natural nucleotides (A, T, G, C) as well as modified nucleotides which comprise at least one modification such as (1) an analog of a purine, (2) an analog of d pyrimidine, or (3) a similar sugar, examples of such modified nucleotides being described for example in PCT application No. WO 95/04 064.
  • a first polynucleotide is considered to be "complementary "of a second polynucleotide when each base of the first nucleotide is paired with the complementary base of the second polynucleotide whose orientation is reversed.
  • the complementary bases are A and T (or a and U), or C and G.
  • variant of a nucleic acid is meant a nucleic acid which differs from one or more bases with respect to the polynucleotide reference.
  • a variant nucleic acid may be of natural origin, such as an allelic variant found naturally, or may also be an unnatural variant obtained for example by mutagenesis techniques.
  • the differences between the reference nucleic acid and the variant nucleic acid are reduced so that the nucleotide sequences of the reference nucleic acid and the variant nucleic acid are very close and, in many regions , identical.
  • the nucleotide modifications present in a variant nucleic acid can be silent, which means that they do not alter the amino acid sequences encoded by said variant nucleic acid.
  • changes in nucleotides in a variant nucleic acid can also result in substitutions, additions, deletions in the polypeptide encoded by the nucleic acid. varying with respect to the peptides encoded by the reference nucleic acid.
  • changes to nucleotides in the coding regions can produce substitutions, conservative or non-conservative in the amino acid sequence.
  • the variant nucleic acids according to the invention encode polypeptides which retain substantially the same biological function or activity as the polypeptide of the reference nucleic acid or else the ability to be recognized by antibodies directed against the polypeptides encoded by l initial nucleic acid.
  • nucleic acids will thus code for mutated forms of polypeptides whose systematic study will make it possible to deduce structure activity relationships from the proteins in question. Knowledge of these mutations in relation to the disease studied is fundamental since it allows us to understand the molecular cause of the pathology.
  • fragment will be understood to mean a reference nucleic acid according to the invention, a nucleotide sequence of reduced length compared to the reference nucleic acid and comprising, on the common part, a nucleotide sequence identical to the nucleic acid of reference.
  • Such a “fragment” of nucleic acid according to the invention may, where appropriate, be included in a larger polynucleotide of which it is constitutive.
  • Such fragments include, or alternatively consist of oligonucleotides of length ranging from 8, 10, 12, 15, 18, 20 to 25, 30, 40, 50, 70, 80, 100, 200, 500, 1000 or 1500 nucleotides of a nucleic acid according to the invention.
  • variant of a polypeptide according to the invention is mainly meant a polypeptide whose amino acid sequence contains one or more substitutions, additions or deletions of at least one amino acid residue, relative to the sequence amino acids of the reference polypeptide, it being understood that the amino acid substitutions can be indifferently conservative. or not conservative.
  • fragment of a polypeptide according to the invention, is meant a polypeptide whose amino acid sequence is shorter than that of the reference polypeptide and which comprises over the entire part common with these reference polypeptides, a sequence in identical amino acids.
  • Such fragments may, if appropriate, be included within a larger polypeptide of which they are part.
  • Such fragments of a polypeptide according to the invention can have a length of 10, 15, 20, 30 to 40, 50, 100, 200 or 300 amino acids.
  • the "percentage of identity" between two nucleotide or amino acid sequences, within the meaning of the present invention, can be determined by comparing two optimally aligned sequences, through a comparison window.
  • the part of the nucleotide or polypeptide sequence in the comparison window can thus include additions or deletions (for example "gaps") with respect to the reference sequence (which does not include these additions or these deletions) so as to obtain an optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which an identical nucleic base or amino acid residue is observed for the two sequences (nucleic or peptide) compared, then by dividing the number of positions at which there is identity between the two bases or amino acid residues by the total number of positions in the comparison window, then multiplying the result by 100 to obtain the percentage of sequence identity.
  • the optimal alignment of the sequences for the comparison can be achieved by computer using known algorithms contained in the package of the company WISCONSIN GENETICS SOFTWARE PACKAGE, GENETICS COMPUTER GROUP (GCG), 575 Science Doctor, Madison, WISCONSIN.
  • the percentage of sequence identity may be carried out using the BLAST software (BLAST versions 1.4.9 of March 1996, BLAST 2.0.4 of February 1998 and BLAST 2.0.6 of September 1998), using only the default parameters (S. F AltschuI et al, J. Mol. Biol. 1990 215: 403-410, S. F AltschuI et al, Nucleic Acids Res. 1997 25: 3389-3402).
  • the query sequence and the databases used can be peptide or nucleic, any combination being possible.
  • hybridization conditions described above are suitable for hybridization under conditions of high stringency, of a nucleic acid molecule of variable length from 20 nucleotides to several hundred nucleotides.
  • hybridization conditions described above can be adapted as a function of the length of the nucleic acid for which hybridization is sought or of the type of labeling chosen, according to techniques known to those skilled in the art.
  • the suitable hybridization conditions can for example be adapted according to the teaching contained in the work of HAMES and HIGGINS (1985) or also in the work of F. AUSUBEL et al (1999).
  • Nucleic acid (s) II was isolated according to the invention a messenger RNA corresponding to a transcript of the gene designated here under the number GS9002S31.
  • the nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 1.
  • sequence SEQ ID No. 1 is 552 nucleotides in length. No sequence identity was found during a search in the GenBank database (Version 110).
  • the analyzes of expression of the transcript of sequence SEQ ID No. 1 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS9002S31 gene was expressed in the fetal brain, liver and placenta. Gene GS910331.
  • Two messenger RNA sequences corresponding to a transcript of the gene designated here under the number GS910331 have been isolated according to the invention.
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 2.
  • sequence SEQ ID No. 2 is 1246 nucleotides in length.
  • sequence SEQ ID No. 3 is 3035 nucleotides in length.
  • the analyzes of expression of the transcript of sequence SEQ ID No. 2 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS310331 gene was expressed in the fetal brain.
  • 83 is 1.65 kb and 2.4 kb in the heart and 1.65 kb in the liver, respectively.
  • This gene is a causal positional candidate for disease caused by dysfunction of the reverse cholesterol flow, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31 -34 on chromosome 9.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS94554 has been isolated according to the invention.
  • the nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 4.
  • sequence SEQ ID No. 4 is 1479 nucleotides in length. No sequence identity was found during a search in the GenBank database (Version 1 10).
  • the expression analyzes of the transcript of sequence SEQ ID No. 4 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS94554 gene was expressed in the fetal brain, the placenta and the liver.
  • an analysis of the expression of the transcript by Northern blot, according to the protocol described in Example 1, using respectively the probe of sequence SEQ ID N c 58, revealed the presence of transcripts in the blot. marketed by the Clontech Company (Ref. N ° 7759-1).
  • the size of the transcripts detected with the sequence probe SEQ ID No. 84 is respectively:
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID N 5.
  • sequence SEQ ID No. 5 is 5169 nucleotides in length. No sequence homology with the sequence SEQ ID N ° 5 was found during a search in the GenBank database (Version 110).
  • the second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID N 6.
  • the sequence SEQ ID No. 6 has a length of 7723 nucleotides. This sequence includes an open reading frame (ORF) going from the nucleotide at position 121 to the nucleotide at position 1517 of the sequence SEQ ID No. 6.
  • the codon for initiating translation begins at the nucleotide at position 132 of the sequence SEQ ID N ° 6.
  • the coding sequence begins at the nucleotide at position 132 and ends at the nucleotide at position 1517 of the sequence SEQ ID N ° 6.
  • SEQ ID No. 6 includes a polyadenylation sequence signal
  • the analyzes of expression of the transcript of sequence SEQ ID No. 5 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the gene GS914739 was expressed in the fetal brain.
  • the size of the transcripts detected with the sequence probe SEQ ID No. 85 is 1 kb in the heart, the liver, the skeletal muscle and the kidney.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • the open reading frame of the nucleotide sequence SEQ ID No. 6 potentially codes for a polypeptide of 461 amino acids in length constituting the sequence SEQ ID No. 129.
  • a sequence identity of approximately 30% was found with in the bases Genpept115, Swissprot38, trEMBL and PIR the following access numbers : AF035360 (homo), AF186461
  • polypeptide of sequence SEQ ID No. 129 is capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier disease, in family deficiencies in HDL, or in a disease genetically linked to the locus 9q31-34 of chromosome 9.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS915574 has been isolated according to the invention.
  • the nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 7.
  • SEQ ID NO: 7 is 1046 nucleotides in length.
  • the analyzes of expression of the transcript of sequence SEQ ID No. 7 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS915574 gene was expressed in the fetal brain, uterus, brain, heart, prostate, fetal liver, liver, placenta, testis and kidney.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS930321 has been isolated according to the invention.
  • the nucleic acid sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 8.
  • sequence SEQ ID No. 8 is 280 nucleotides in length. No sequence identity was found during a search in the GenBank database (Version 1 10).
  • the analyzes of expression of the transcript of sequence SEQ ID No. 8 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS930321 gene was expressed in the fetal brain, liver and heart.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • Nucleic acid (s) II was isolated according to the invention a messenger RNA corresponding to a transcript of the gene designated here under the number GS931311.
  • the nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 9.
  • sequence SEQ ID No. 9 is 479 nucleotides in length. This sequence includes a partial open reading frame (ORF) going from the nucleotide in position 3 to the nucleotide in position 98 of the sequence SEQ ID NO 9
  • ORF partial open reading frame
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or to a disease genetically linked to locus 9q31 -34 of the chromosome 9.
  • Polypeptide encoded by the nucleic acid of sequence SEQ ID NO 9 The open partial reading frame of the nucleic acid sequence SEQ ID No. 9 potentially codes for a polypeptide of 32 amino acids in length constituting the sequence SEQ ID No. 130.
  • polypeptide of sequence SEQ ID No. 130 is capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier's disease, in family deficiencies in HDL, or in a disease genetically linked to the locus 9q31-34 of chromosome 9.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS934660 has been isolated according to the invention.
  • the nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 10.
  • SEQ ID NO: 10 is 2599 nucleotides in length.
  • the analyzes of expression of the transcript of sequence SEQ ID No. 10 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS934660 gene was expressed in the fetal brain.
  • the size of the transcripts detected with the sequence probe SEQ ID No. 86 is respectively: - 1 kb, 2 kb, 3 kb and 7.5 kb in the placenta;
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or to a disease genetically linked to locus 9q31 -34 of the chromosome 9.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS938315 has been isolated according to the invention.
  • the nucleic acid sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 1 1.
  • sequence SEQ ID No. 11 11 is 222 nucleotides in length.
  • Nucleic acid (s) II was isolated according to the invention two messenger RNAs corresponding to a transcript of the gene designated here under the number GS93953.
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 12.
  • SEQ ID NO: 12 is 3422 nucleotides in length.
  • the second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No 13.
  • the sequence SEQ ID No 13 has a length of 5791 nucleotides.
  • ORF open reading frame
  • the expression analyzes of the transcript of sequence SEQ ID No. 12 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues have shown that the GS93953 gene is expressed in the fetal brain.
  • the size of the transcripts detected with the sequence probe SEQ ID N c 87 is 8 kb in the heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • the partial open reading frame of the nucleic acid sequence SEQ ID No. 13 potentially codes for a polypeptide of 183 amino acids in length constituting the sequence SEQ ID No. 131.
  • polypeptide of sequence SEQ ID No. 131 is capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier's disease, in deficiencies in HDL, or in a disease genetically linked to locus 9q31-34 of chromosome 9.
  • RNAs corresponding to a transcript of the gene designated here under the number GS939874 have been isolated according to the invention.
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 14.
  • SEQ ID NO: 14 is 2615 nucleotides in length.
  • the second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 15.
  • the sequence SEQ ID No. 15 has a length of 2551 nucleotides.
  • It comprises an open reading frame going from the nucleotide at position 50 to the nucleotide at position 958 and a coding sequence going from the nucleotide at position 67 to the nucleotide at position 958.
  • the analyzes of expression of the transcript of sequence SEQ ID No. 14 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS939874 gene was expressed in the fetal brain, uterus, brain, heart, prostate, fetal liver, liver, placenta, testis and kidney.
  • This gene is a causal positional candidate for disease caused by dysfunction of the reverse cholesterol flow, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 on chromosome 9.
  • the open reading frame of the nucleic acid of sequence SEQ ID No 15 potentially codes for a polypeptide of 291 amino acids in length constituting the sequence SEQ ID No 132.
  • polypeptide of sequence SEQ ID No. 132 is capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier's disease, in family deficiencies in HDL, or in a disease genetically linked to the locus 9q31-34 of chromosome 9.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS91 1370 has been isolated according to the invention.
  • the nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 16.
  • the sequence SEQ ID No. 16 is 775 nucleotides in length. This sequence includes an open partial reading frame (ORF) going from the nucleotide in position 1 to the nucleotide in position 144 of the sequence SEQ ID No. 16.
  • ORF open partial reading frame
  • the analyzes of expression of the transcript of sequence SEQ ID No. 16 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS911370 gene was expressed in the fetal brain.
  • the size of the transcripts detected with the sequence probe SEQ ID No. 88 as well as with the sequence probe SEQ ID No. 89 is 7.4 kb in the pancreas.
  • This gene is a causal positional candidate for disease caused by dysfunction of the reverse cholesterol flow, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 on chromosome 9.
  • the open partial reading frame of the nucleic acid sequence SEQ ID No. 16 potentially codes for a polypeptide of 48 amino acids in length constituting the sequence SEQ ID No. 133. Homologies observed at the level of the protein sequence:
  • This potential ORF (48 aa) has an identity on 33 aa (position 16-48 aa) with the transport protein under beta unit of the human Sec61 complex whose sequences are sp
  • the protein complex sec61 is a central component of the cellular machinery for translocation of nascent proteins in the endoplasmic reticulum.
  • the GS911370 gene could therefore be, due to its homology with the beta subunit of the sec61 complex, a gene coding for a new component of this machinery. Consequently its possible role in the translocation of proteins and consequently in the trafficking of proteins involved in the efflux mechanism of cholesterol makes it a gene of interest in the study of the deficit observed in Tangier / FHD patients.
  • polypeptide of sequence SEQ ID No. 133 is thus capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier's disease or of family deficiencies in HDL.
  • polypeptide of sequence SEQ ID N133 is therefore capable of intervening in an important step involved in the reverse transport of cholesterol by HDL.
  • the polypeptide of sequence SEQ ID No. 133 is also capable of intervening in a disease genetically linked to the locus
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS913920 has been isolated according to the invention.
  • a first sequence was isolated and characterized; it is the nucleic sequence of the cDNA constituting the sequence SEQ ID No. 17.
  • the sequence SEQ ID No. 17 is 491 nucleotides in length.
  • the expression analyzes of the transcript of sequence SEQ ID No. 17 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS913920 gene was expressed in the liver and the heart. From the sequence SEQ ID No. 17, two nucleotide primers of sequences SEQ ID No. 102 and SEQ ID No. 103 respectively have been synthesized. These nucleotide primers made it possible to amplify a cDNA of the GS913920 gene constituting the sequence SEQ ID No. 18.
  • the amplification reactions are carried out under the following conditions, applicable to all the candidate genes according to the invention for which specific primers are described and used to isolate sequences of the transcript of interest:
  • Each PCR reaction is carried out with 400 ⁇ M of each dNTP, 0.5 ⁇ M of each primer, 2.5 mM of MgCI2, 50 ng of DNA or approximately 25 ng of cDNA and 2 units of Thermus aquatic ⁇ s (Taq) DNA polymerase (Ampli Taq Gold; Perkin Elmer) in the presence of its buffer.
  • the reactions are carried out in 96-well microplates, in 9700 thermocyclers (Perkin Elmer). After a first denaturation at 94 ° C for 10 min, a program of 30 cycles is applied: denaturation of 30 s. at 94 ° C, 30 s hybridization.
  • the nucleic sequence SEQ ID No. 18 is 293 nucleotides in length. It includes a partial open reading frame (ORF) going from the nucleotide in position 227 to the nucleotide in position 293. This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly of the disease de Tangier or deficiencies in HDL, or to a disease genetically linked to locus 9q31-34 on chromosome 9.
  • ORF partial open reading frame
  • the open reading frame of the nucleic acid sequence SEQ ID No. 18 potentially codes for a polypeptide of 22 amino acids in length constituting the sequence SEQ ID No. 134.
  • TrEMBL (translation of Genbank v115), TrEMBL (SP-TrEMBL, August version
  • polypeptide of sequence SEQ ID No. 134 is capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier's disease or of family deficiencies in HDL.
  • polypeptide of sequence SEQ ID No. 134 is therefore capable of intervening in an important step involved in the reverse transport of cholesterol by HDL.
  • polypeptide of sequence SEQ ID No. 134 is also capable of intervening in a disease genetically linked to locus 9q31-34 of chromosome 9.
  • RNAs corresponding to a transcript of the gene designated here under the number GS91437 have been isolated according to the invention.
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 19.
  • the sequence SEQ ID No. 19 is 2442 nucleotides in length.
  • This sequence includes a partial open reading frame (ORF) going from the nucleotide in position 2 to the nucleotide in position 286 of the sequence SEQ ID No. 19. No sequence identity was found during a search in the GenBank database (Version 1 10).
  • the second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 20.
  • SEQ ID NO: 20 is 4608 nucleotides in length. It includes a partial open reading frame (ORF) going from the nucleotide in position 1 to the nucleotide in position 327.
  • ORF partial open reading frame
  • the analyzes of expression of the transcript of sequence SEQ ID No. 19 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS91437 gene was expressed in the fetal brain, liver, heart, prostate, placenta, uterus, testis, kidney, skeletal muscle.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or deficiencies. in HDL, or to a disease genetically linked to locus 9q31-34 on chromosome 9.
  • the open partial reading frame of the nucleic acid sequence SEQ ID No 19 potentially codes for a polypeptide of 95 amino acids in length constituting the sequence SEQ ID No 135.
  • polypeptide of sequence SEQ ID No. 135 is capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier's disease, in family deficiencies in HDL, or in a disease genetically linked to the locus 9q31-34 of chromosome 9.
  • the open reading frame (ORF) contained in the nucleic acid of sequence SEQ ID No. 20 potentially codes for a polypeptide of 108 amino acids constituting the sequence SEQ ID No. 136.
  • sequence homology was found with the databases Swissprot (Version 38), Genpept (Version 1 15), PIR (Version 62, September 1999) and trEMBL (Version of August 1999).
  • the polypeptide of sequence SEQ ID No. 136 is capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier's disease, in family deficiencies in HDL, or in a disease genetically linked to the locus 9q31-34 of chromosome 9.
  • RNAs corresponding to a transcript of the gene designated here under the no. GS91507 have been isolated according to the invention.
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 21.
  • SEQ ID NO: 21 is 1627 nucleotides in length. This sequence includes a partial open reading frame (ORF) going from the nucleotide in position 1 to the nucleotide in position 640 of the sequence SEQ ID No. 21.
  • ORF partial open reading frame
  • SEQ ID NO: 22 is 2333 nucleotides in length. It includes a complete open reading frame (ORF) ranging from the nucleotide at position 368 to the nucleotide at position 1348. The start of the translation initiation codon is located on the nucleotide at position 371 of the sequence SEQ ID No. 22. The coding sequence begins at the nucleotide at position 371 and ends at the nucleotide at position 1348.
  • ORF complete open reading frame
  • sequence SEQ ID N ° 22 has a homology with the following sequence referenced in the following GenBank database (Version 1 16): 99% nucleic identity over 2316 bp (position 115 to 2420 bp) with Homo sapiens cDNA FLJ20300 fis, clone HEP06465 (2331 bp). Accession AK000307
  • the analyzes of expression of the transcript of sequence SEQ ID No. 21 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS91507 gene was expressed in the fetal brain.
  • the size of the transcripts detected with the sequence probe SEQ ID No. 90 is respectively:
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction in the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL.
  • Polypeptide encoded by the nucleic acid of sequence SEQ ID NO 21 The open reading frame of the nucleic acid sequence
  • SEQ ID No. 21 potentially codes for a polypeptide of 213 amino acids in length constituting the sequence SEQ ID No. 137. Homologies observed at the protein sequence level: This potential ORF of 213 aa has a domain having motif type homologies with different proteins such as: • 29% homology on 155 aa (position 4-158 aa) with Swissprot sequences (version 36, last update of May 3, 1999) sp
  • Genpept (translation of Genbank v1 10 and 1 1 1, last update May 7, 1999)) would show annotations with potential proteins of the type "phosphatidic acid phosphatase"
  • Genpept sequences (translation of Genbank v1 10 and 1 1 1, last update on May 7, 1999): gi
  • the open reading frame of the nucleic acid sequence SEQ ID No 22 potentially codes for a polypeptide of 325 amino acids in length constituting the sequence SEQ ID No 138. Homologies observed at the level of the protein sequence
  • the GS91507 gene is of interest for the functional study of Tangier's disease and FHD in the sense that it is both a positional candidate and a gene coding for a new protein whose functions putatives (phosphatidic acid phosphatase) could play a role in the intracellular signaling cascade linked to the efflux of intracellular cholesterol mediated by HDL particles, involved in Tangier disease and familial HDL deficiency (FHD).
  • the polypeptides of sequence SEQ ID No. 137 and 138 is capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier's disease, in family deficiencies in HDL, or in a disease genetically linked to the locus 9q31. -34 of chromosome 9.
  • RNAs corresponding to a transcript of the gene designated here under the number GS915231 have been isolated according to the invention.
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 23.
  • SEQ ID NO: 23 is 2764 nucleotides in length.
  • This sequence includes a partial open reading frame (ORF) going from the nucleotide in position 3 to the nucleotide in position 1220 of the sequence SEQ ID No. 23.
  • ORF partial open reading frame
  • the second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 24.
  • the sequence SEQ ID No. 24 has a length of 3228 nucleotides. It comprises an open reading frame (ORF) going from the nucleotide at position 37 to the nucleotide at position 1304.
  • the coding sequence begins at the nucleotide at position 49 and ends at the nucleotide at position 1304 of the sequence SEQ ID No. 24.
  • the start of the translation initiation codon is located on the nucleotide at position 49.
  • the analyzes of expression of the transcript of sequence SEQ ID No. 23 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS915231 gene was expressed in the fetal brain.
  • the size of the transcripts detected with the sequence probe SEQ ID No. 91 is respectively:
  • skeletal muscle - 1, 3 kb, 2 kb, 4 kb and 4.4 kb in the liver and the kidney;
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • the open partial reading frame of the nucleic acid sequence SEQ ID No 23 potentially codes for a polypeptide of 406 amino acids in length constituting the sequence SEQ ID No 139. Homologies observed at the level of the protein sequence:
  • Genpept sequences (translation of Genbank v1 10 and 1 1 1, last update May 7, 1999): gi: 2731377 is the protein translation of the sequence gb
  • the open reading frame (ORF) of the nucleic acid sequence SEQ ID No. 24 potentially codes for a polypeptide of 422 amino acids in length constituting the sequence SEQ ID No. 140.
  • the following sequence homologies have been found: Homologies observed at the level of the protein sequence:
  • Genpept: gi: 2731377 is the protein translation of the gb sequence
  • Putative function The ORF of 422 amino acids has homologies with different putative enzymatic proteins involved in the dehydrogenation mechanisms of sterols in several organisms: man, mouse, E. coli, S. cerevisiae, C. elegans.
  • the GS15231 gene codes for a protein of interest by virtue of its possible function in the defect of intracellular cholesterol trafficking in Tangier / FHD patients, which reinforces the interest of studying the GS15231 gene.
  • the GS15231 gene is, by its location, a positional candidate gene for the study and characterization of the genetic defect observed in Tangier's disease or the familial defect in HDL.
  • polypeptides of sequences SEQ ID N ° 139 and 140 are capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier's disease, in family deficiencies in HDL, or in a disease genetically linked to the locus 9q31. -34 of chromosome 9. Gene GS915528.
  • RNAs corresponding to a transcript of the gene designated here under the number GS915528 have been isolated according to the invention.
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 25.
  • sequence SEQ ID No. 25 is 3106 nucleotides in length.
  • This sequence includes a partial open reading frame (ORF) going from the nucleotide in position 1 to the nucleotide in position 1272 of the sequence SEQ ID No. 25.
  • ORF partial open reading frame
  • the second nucleic sequence of the ADNC corresponding to this transcript constitutes the sequence SEQ ID No. 26.
  • the sequence SEQ ID No. 26 has a length of 3313 nucleotides.
  • liver the liver, prostate, heart, placenta, uterus, testis and brain.
  • the size of the transcripts detected with the sequence probe SEQ ID No. 92 is respectively: - 1.9 kb, 3.2 kb and 3.8 kb in the pancreas; - 1 kb, 1.9 kb and 3.8 kb in the heart;
  • This gene is a causal positional candidate for disease caused by dysfunction of the reverse cholesterol flow, and more
  • SEQ ID No. 25 potentially codes for a polypeptide of 424 amino acids in length constituting the sequence SEQ ID No. 141.
  • This ORF of 424 aa has motif type homologies with conserved domains of different tyrosine phosphatase proteins in different protein databases such as:
  • the open reading frame of the nucleic acid sequence SEQ ID No. 26 potentially codes for a polypeptide of 455 amino acids in length constituting the sequence SEQ ID No. 142.
  • This ORF of 455 aa has motif type homologies with conserved domains of different proteins tyrosine phosphatase in different protein databases such as. • 50% homology on 374 aa (position 24-397 aa) with SP-trEMBL: sp
  • mice and Swissprot sp
  • the GS915528 gene is of interest for the functional study of Tangier's disease and FHD in the sense that it is both a positional candidate and a gene coding for a new protein whose functions putatives (tyrosine phosphatase) could play a role in the intracellular signaling cascade linked to the efflux of intracellular cholesterol mediated by HDL particles, implicated in Tangier disease and familial HDL deficiency (FHD).
  • polypeptides of sequence SEQ ID No. 141 and 142 are capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier's disease, in family deficiencies in HDL, or in a disease genetically linked to the locus 9q31. -34 of chromosome 9.
  • RNAs corresponding to a transcript of the gene designated here under the number GS99817 have been isolated according to the invention.
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 27.
  • sequence SEQ ID No. 27 is 1539 nucleotides in length.
  • This sequence includes a partial open reading frame (ORF) going from the nucleotide in position 3 to the nucleotide in position 698 of the sequence SEQ ID No. 27. No sequence identity was found during a search in the GenBank database (Version 1 10).
  • the second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 28.
  • SEQ ID NO: 28 is 3404 nucleotides in length. It includes a partial open reading frame (ORF) ranging from the nucleotide in position 1 to the nucleotide in position 792 of the sequence SEQ ID No. 28.
  • ORF partial open reading frame
  • the analyzes of expression of the transcript of sequence SEQ ID No. 27 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the gene GS99817 was expressed in the fetal brain.
  • the size of the transcripts detected with the sequence probe SEQ ID No. 93 is respectively: - 1.5 kb, 2 kb and 4.4 kb in the heart and the brain;
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • the open reading frame of the nucleic acid sequence SEQ ID No. 27 potentially codes for a polypeptide of 232 amino acids in length constituting the sequence SEQ ID No. 143. Homologies observed at the level of the protein sequence:
  • Genpept sequences (translation of Genbank v110 and 111, last update on May 7, 1999): gi
  • the open reading frame of the nucleic acid sequence SEQ ID No 28 potentially codes for a polypeptide of 263 amino acids in length constituting the sequence SEQ ID No 144. Homologies have been observed between the sequence SEQ ID No 144 and the following sequences:
  • This gene is a candidate for the study of Tangier disease / FHD due to the chromosomal location.
  • polypeptides of sequences SEQ ID No. 143 and 144 are capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier's disease, in family deficiencies in HDL, or in a disease genetically linked to the locus 9q31. -34 of chromosome 9.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS916229 has been isolated according to the invention.
  • the nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 29.
  • sequence SEQ ID No. 29 is 792 nucleotides in length. This sequence includes an open reading frame (ORF) going from the nucleotide in position 1 to the nucleotide in position 203 of the sequence SEQ ID No. 29.
  • ORF open reading frame
  • the expression analyzes of the transcript of sequence SEQ ID No. 29 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS916229 gene was expressed in the fetal brain, the liver, the brain, the heart, the prostate, the placenta, the fetal liver, the uterus, the testicle and the kidney.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • the open reading frame of the nucleic acid sequence SEQ ID No. 29 potentially codes for a polypeptide of 68 amino acids in length constituting the sequence SEQ ID No. 145.
  • polypeptide of sequence SEQ ID No. 145 is capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier disease, in family deficiencies in HDL, or in a disease genetically linked to the locus 9q31-34 of chromosome 9.
  • GS92544 Nucleic acid (s) Three messenger RNAs corresponding to a long transcript and two short transcripts of the gene designated here under the no. GS92544 have been isolated according to the invention.
  • the nucleic sequence of the cDNA corresponding to the long transcript constitutes the sequence SEQ ID No. 30.
  • SEQ ID NO: 30 is 2733 nucleotides in length.
  • This sequence includes a partial open reading frame (ORF) going from the nucleotide in position 1 to the nucleotide in position 2160 of the sequence SEQ ID No. 30.
  • ORF partial open reading frame
  • the first nucleic sequence of the cDNA corresponding to the short transcript constitutes the sequence SEQ ID No. 31.
  • SEQ ID NO: 31 is 2694 nucleotides in length.
  • This sequence includes a partial open reading frame (ORF) going from the nucleotide in position 1 to the nucleotide in position 2121 of the sequence SEQ ID No. 31.
  • ORF partial open reading frame
  • the second nucleic sequence of the cDNA corresponding to the second short transcript constitutes the sequence SEQ ID No. 32.
  • SEQ ID NO: 32 is 2765 nucleotides in length. It includes a complete open reading frame going from the nucleotide in position 56 to the nucleotide in position 2287 of the sequence SEQ ID No. 32.
  • the coding sequence begins at the nucleotide in position 410 and ends at the nucleotide in position 2160 of the sequence SEQ ID No. 32 ..
  • the translation initiation codon begins on the nucleotide at position 410 of the sequence SEQ ID No. 32.
  • the analyzes of expression of the long transcript and of the short transcript were carried out by RT PCR, as described in Example 1. These analyzes carried out from polyA + RNA from different tissues made it possible to show that the GS92544 gene was expressed in the fetal brain.
  • transcripts detected with the sequence probe SEQ ID No. 94 are 4 kb and 6 kb respectively in the placenta.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • the open reading frame of the nucleic acid sequence SEQ ID No. 30 (long transcript) potentially codes for a polypeptide of 720 amino acids in length constituting the sequence SEQ ID No. 146.
  • SEQ ID No. 31 (short transcript) potentially codes for a polypeptide of 707 amino acids in length constituting the sequence SEQ ID No. 147.
  • Polypeptide encoded by the nucleic acid of sequence SEQ ID No. 32 The open reading frame of the nucleic acid sequence SEQ ID No. 32.
  • ID No. 32 potentially codes for a polypeptide of 713 amino acids in length constituting the sequence SEQ ID No. 148.
  • the sequence SEQ ID No. 148 has characteristic motifs such as 3 EGF domains including 2 calcium binding, a tyrosine phosphatase site, a N-terminal hydrophobic domain; Numerous glycosylation sites; 2 camp phosphorylation sites; 2 sites Asp hydroxylation.
  • GS92544 is putatively a membrane associated protein like uromodulin which is a protein anchored by means of a glycosylphosphatidylinositol (GPI).
  • GPI glycosylphosphatidylinositol
  • polypeptides of sequences SEQ ID No. 146, 147 and 148 are capable of intervening in the regulation of the flow of cholesterol, and more particularly Tangier's disease, in familial HDL deficiencies, or in a disease genetically linked to locus 9q31-34 on chromosome 9.
  • RNAs corresponding to a transcript of the gene designated here under the number GS930824 have been isolated according to the invention.
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 33.
  • sequence SEQ ID No. 33 is 4745 nucleotides in length.
  • This sequence includes a partial open reading frame (ORF) going from the nucleotide in position 2 to the nucleotide in position 514 of the sequence SEQ ID No. 33. Homologies observed at the nucleotide sequence level:
  • Genbank AF1 15435, rat syntaxin 17 on 510 bp (position 22-531 bp).
  • SEQ ID No. 34 The second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 34.
  • SEQ ID NO: 34 is 5241 nucleotides in length.
  • the coding sequence begins at the nucleotide at position 105 and ends at the nucleotide at position 1013 of the sequence SEQ ID No. 34.
  • the translation initiation codon begins on the nucleotide at position 105 of the sequence SEQ ID No. 34.
  • sequence SEQ ID No. 34 has homologies with sequences referenced in the GenBank database (Version 1 16). These are the following homologies:
  • the analyzes of expression of the transcript of sequence SEQ ID No. 33 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS930824 gene was expressed in the fetal brain, liver, brain, heart, prostate, placenta, fetal liver, uterus, testis, kidney and skeletal muscle.
  • the size of the transcripts detected with the sequence probe SEQ ID No. 95 is respectively:
  • the size of the transcripts detected with the sequence probe SEQ ID No. 96 is respectively: - 1.35 kb, 2.4 kb, 3.5 kb and 10 kb in the placenta; - 1.35 kb and 2.4 kb in the pancreas, the kidney and the liver; - 1.35 kb in the lung;
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction in the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL.
  • the open reading frame of the nucleic acid sequence SEQ ID No. 33 potentially codes for a polypeptide of 170 amino acids in length constituting the sequence SEQ ID No. 149.
  • No significant homology with BLAST was found with the sequences referenced in the Swissprot (version 36, last update May 3, 1999) and PRODOM: databases (homologous domains detected in Swissprot, version 34.2, November 1997).
  • gi4206161 is the protein translation of the gene coding for rat syntaxin 17 .
  • Genpept (translation of Genbank v110 and 111, last update May 7, 1999):
  • the open reading frame of the sequence SEQ ID No. 34 potentially codes for a polypeptide of 318 amino acids constituting the sequence SEQ ID No. 150.
  • Genpept: gi4206161 is the protein translation of the gene coding for rat syntaxin 17.
  • the polypeptide of sequence SEQ ID No. 150 has a motif characteristic of a syntaxin between the nucleotides at positions 1 and 243 of the open reading frame.
  • polypeptides of sequence SEQ ID Nos. 149 and 150 therefore resemble a protein of the syntaxin family involved in intracellular vesicular transport. This underlying mechanism by specific addressing mediated by these proteins is of interest with respect to Tangier / FHD disease which is described by a defect in the mechanism of transport and translocation of pools of intracellular cholesterol towards acceptor HDL particles.
  • the polypeptides of sequence SEQ ID Nos. 149 and 150 are therefore capable of intervening in an important step involved in the reverse transport of cholesterol by HDL.
  • polypeptides of sequences SEQ ID Nos. 149 and 150 are also capable of intervening in a disease genetically linked to locus 9q31-34 of chromosome 9.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS93382 has been isolated according to the invention.
  • the nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 35.
  • sequence SEQ ID No. 35 is 3014 nucleotides in length.
  • This sequence includes a partial open reading frame (ORF) going from the nucleotide in position 3 to the nucleotide in position 371 of the sequence SEQ ID No. 35.
  • ORF partial open reading frame
  • the expression analyzes of the transcript of sequence SEQ ID No. 35 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS93382 gene was expressed in the fetal brain.
  • the size of the transcripts detected with the sequence probe SEQ ID No. 97 is respectively: - 2 kb and 7.5 kb in the brain;
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to the locus.
  • the open reading frame of the nucleic acid sequence SEQ ID No 35 potentially codes for a polypeptide of 123 amino acids in length constituting the sequence SEQ ID No 151.
  • polypeptide of sequence SEQ ID No. 151 is capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier's disease, in deficiencies in HDL, or in a disease genetically linked to locus 9q31-34 of chromosome 9.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS946300 has been isolated according to the invention.
  • the nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 36.
  • SEQ ID NO: 36 is 1575 nucleotides in length.
  • This sequence includes a partial open reading frame (ORF) going from the nucleotide in position 3 to the nucleotide in position 176 of the sequence SEQ ID No. 36. No sequence identity was found during a search in the GenBank database (Version 110).
  • the analyzes of expression of the transcript of sequence SEQ ID No. 36 were carried out by RT PCR, as described in Example 1. These analyzes carried out from polyA + RNA from different tissues made it possible to show that the GS946300 gene was expressed in the fetal brain, liver, brain, heart, prostate, placenta, fetal liver, uterus, testicle and kidney.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • the open reading frame of the nucleic acid sequence SEQ ID No. 36 potentially codes for a polypeptide of 58 amino acids in length constituting the sequence SEQ ID No. 152.
  • No significant homology with BLAST was found with the sequences referenced in the Swissprot (version 36, last update May 3, 1999) and PRODOM: databases (homologous domains detected in Swissprot, version 34.2, November 1997).
  • the polypeptide of sequence SEQ ID No. 152 is capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier's disease or of family deficiencies in HDL.
  • polypeptide of sequence SEQ ID No. 152 is therefore capable of intervening in an important step involved in the reverse transport of cholesterol by HDL.
  • RNAs corresponding to a long transcript and a short transcript of the gene designated here under the no. GS937345 have been isolated according to the invention.
  • a first nucleic sequence of the cDNA corresponding to the long transcript was isolated and constitutes the sequence SEQ ID No. 37.
  • sequence SEQ ID No. 37 is 1607 nucleotides in length.
  • This sequence includes a partial open reading frame (ORF) going from the nucleotide in position 2 to the nucleotide in position 109 of the sequence SEQ ID No. 37.
  • ORF partial open reading frame
  • two primers of respective sequences SEQ ID No. 104 and 105 were synthesized and made it possible to amplify a cDNA from a bank of polyA + mRNA of different human tissues marketed by the Clontech company.
  • SEQ ID Nos. 104 and 105 constitutes the sequence SEQ ID No. 38.
  • the sequence SEQ ID No. 38 is 1161 nucleotides in length.
  • the nucleic sequence of the cDNA corresponding to the short transcript constitutes the sequence SEQ ID No. 39.
  • SEQ ID NO: 39 is 1332 nucleotides in length.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • the open partial reading frame of the long transcript of nucleic acid sequence SEQ ID No. 37 potentially codes for a polypeptide of 36 amino acids in length constituting the sequence SEQ ID No. 153.
  • RNAs corresponding to a long transcript and a short transcript of the gene designated here under the no. GS99556 have been isolated according to the invention.
  • the nucleic sequence of the cDNA corresponding to the long transcript constitutes the sequence SEQ ID No. 40.
  • SEQ ID NO: 40 is 10419 nucleotides in length.
  • This sequence includes a partial open reading frame (ORF) ranging from the nucleotide in position 2 to the nucleotide in position 1954 of the sequence SEQ ID No. 40.
  • the initiation codon (ATG) begins at the nucleotide at position 29 of the long transcript of sequence SEQ ID No. 40.
  • the nucleic sequence of the cDNA corresponding to the short transcript constitutes the sequence SEQ ID No. 41.
  • the sequence SEQ ID No. 41 is 1813 nucleotides in length.
  • the analyzes of expression of the transcript of sequence SEQ ID No. 40 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS99556 gene was expressed in the fetal brain, liver, brain, heart, prostate, placenta and fetal liver.
  • the size of the transcripts detected with the sequence probe SEQ ID No. 98 is respectively:
  • the size of the transcripts detected with the sequence probe SEQ ID No. 99 is respectively:
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • the open partial reading frame of the nucleic acid sequence SEQ ID No. 40 potentially codes for a polypeptide of 651 amino acids in length constituting the sequence SEQ ID No. 154. Homologies observed at the protein sequence level: This ORF has homologies with Genpept sequences: (translation of Genbank v110 and 111, last updated on May 7, 1999) and TrEMBL (SP-TrEMBL, version 7, November 1998 ) (BlastX translation of the following sequences):
  • This gene is a candidate for the study of Tangier disease / FHD due to the chromosomal location.
  • polypeptide of sequence SEQ ID No. 154 is capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier's disease, in family deficiencies in HDL, or in a disease genetically linked to the locus 9q31-34 of chromosome 9.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS96663 has been isolated according to the invention.
  • Six nucleic acid sequences representative of this transcript have been determined: The first partial nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 42.
  • SEQ ID NO: 42 is 1377 nucleotides in length.
  • the second partial nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 43.
  • sequence SEQ ID No. 43 is 452 nucleotides in length.
  • the third partial nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 44.
  • the sequence SEQ ID No. 44 is 562 nucleotides in length.
  • the fourth partial nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 45.
  • SEQ ID NO: 45 is 1766 nucleotides in length. No sequence identity was found during a search in the GenBank database (Version 110).
  • nucleotide primer of sequence SEQ ID No. 108 was synthesized.
  • sequence SEQ ID No. 45 From the sequence SEQ ID No. 45, two nucleotide primers were synthesized, the primers of sequences SEQ ID No. 109 and 110 respectively.
  • the fifth nucleic sequence corresponding to the transcript of the GS96663 gene constitutes the sequence SEQ ID No. 46.
  • the nucleic sequence SEQ ID No. 46 is 601 nucleotides in length.
  • the sixth nucleic sequence corresponding to the transcript of the GS96663 gene constitutes the sequence SEQ ID No. 47.
  • the sequence SEQ ID No. 47 The sequence SEQ ID No. 47.
  • ID No. 47 is 3706 nucleotides in length. It includes an open partial reading frame going from the nucleotide in position 1 to the nucleotide in position 3202 of the sequence SEQ ID No. 47.
  • the analyzes of expression of the transcript of the sequences SEQ ID Nos. 42 to 47 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GSS96663 gene was expressed in the fetal brain, liver, brain, heart, prostate, placenta, fetal liver, uterus, testis, kidney and skeletal muscle.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction in the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL.
  • the open partial reading frame of the nucleic acid sequence SEQ ID No. 47 potentially codes for a polypeptide of 1066 amino acids in length which constitutes the sequence SEQ ID No. 155.
  • P-selectin and E-selectin sp sequences
  • lem2_human e-selectin precursor endothelial leukocyte adhesion molecule 1 (elam-1) (leukocyte- endothelial cell adhesion molecule 2) (Iecam2) (cd62e) and sp
  • lem3_human p-selectin precursor granule membrane protein 140) (gmp-140) (padgem) (cd62p) (leukocyte-endothelial cell adhesion molecule 3) (Iecam3) and with non-redundant pir: pir
  • a30359 p- selectin precursor - human .. 24% homology on 637 AA with the sequence g183391 (M25322) granule membrane protein-140 (GMP-140) precursor [Homo sapiens], Length 830
  • the GS96663 gene product by virtue of its amino acid homology with human P-selectin and E-selectin precursors, is related to this class of membrane proteins. Its role in the efflux of intracellular cholesterol, mediated by membrane proteins is not excluded, especially as the gene is located in the genetic interval defined by positional cloning.
  • polypeptide of sequence SEQ ID No. 155 is capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier's disease, in family deficiencies in HDL, or in a disease genetically linked to the locus 9q31-34 of chromosome 9.
  • Nucleic acid (s) II was isolated according to the invention a messenger RNA corresponding to a transcript of the gene designated here under the number GS941675. Two nucleic acid sequences are representative of this transcript:
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 48.
  • SEQ ID NO: 48 is 373 nucleotides in length.
  • the second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 49.
  • sequence SEQ ID No. 49 is 459 nucleotides in length.
  • the third nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 50.
  • sequence SEQ ID No. 50 is 2575 nucleotides in length Homologies between the sequence SEQ ID No. 50 and sequences referenced in the GenBank database (Version 1 16) were observed. These homologies are as follows:
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to the locus.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS929341 has been isolated according to the invention. Two nucleic acid sequences representative of this transcript have been determined. The first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 51.
  • sequence SEQ ID No. 51 is 231 nucleotides in length.
  • the second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 52.
  • sequence SEQ ID No. 52 is 344 nucleotides in length. This sequence includes a partial open reading frame (ORF) going from the nucleotide in position 3 to the nucleotide in position 131 of the sequence SEQ ID No. 52. No identity with the sequences SEQ ID N ° 51 and 52 was found during a search in the GenBank database (Version 110).
  • the third nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 53.
  • sequence SEQ ID No. 53 is 402 nucleotides in length. This sequence includes a partial open reading frame (ORF) ranging from the nucleotide in position 1 to the nucleotide in position 188 of the sequence SEQ ID No. 53.
  • ORF partial open reading frame
  • Nos. 51 and 52 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS929341 gene was expressed in the fetal brain, the liver , brain, heart, prostate, placenta, fetal liver, uterus, testis, kidney, skeletal muscle and lung.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to the locus.
  • the open reading frame of the nucleic acid sequence SEQ ID No 52 potentially codes for a polypeptide of 43 amino acids in length constituting the sequence SEQ ID No 156. No significant homology with BLAST was found with the sequences referenced in the Swissprot databases
  • the open reading frame of the nucleic acid sequence SEQ ID No. 53 potentially codes for a polypeptide of 61 amino acids in length constituting the sequence SEQ ID No. 157.
  • polypeptides of sequences SEQ ID N ° 156 and 157 are capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier's disease, in family deficiencies in HDL, or in a disease genetically linked to the locus 9q31 -34 of chromosome 9.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS915742 has been isolated according to the invention. Three nucleic acid sequences representative of this transcript have been determined.
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 54.
  • SEQ ID NO: 54 is 228 nucleotides in length.
  • the second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 55.
  • sequence SEQ ID No. 55 is 270 nucleotides in length.
  • the third nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 56.
  • sequence SEQ ID No. 56 is 1130 nucleotides in length.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS913018 has been isolated according to the invention. Two sequences representative of this transcript are shown below.
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 57.
  • the sequence SEQ ID No. 57 is 463 nucleotides in length.
  • the second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 58.
  • the sequence SEQ ID No. 58 is 289 nucleotides in length.
  • sequence SEQ ID No. 57 From the sequence SEQ ID No. 57, two nucleotide primers were synthesized, the primers of sequences SEQ ID No. 111 and 112 respectively.
  • sequence SEQ ID No. 58 From the sequence SEQ ID No. 58, two nucleotide primers were synthesized, the primers of sequences SEQ ID No. 113 and 114. respectively.
  • the primers of sequences SEQ ID No. 111 to 114 made it possible to amplify a cDNA , from a polyA + mRNA library of different human tissues sold by the Clontech Company, a third nucleic sequence of the cDNA corresponding to the transcript of the GS913018 gene.
  • the third nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 59.
  • sequence SEQ ID No. 59 is 1542 nucleotides in length.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS911742 has been isolated according to the invention. Three representative sequences of this transcript have been determined. The first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 60.
  • sequence SEQ ID No. 60 is 1417 nucleotides in length.
  • the second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 61.
  • SEQ ID NO: 61 is 696 nucleotides in length.
  • the sequence SEQ ID No. 62 is 2702 nucleotides in length. This sequence includes a partial open reading frame (ORF) going from the nucleotide in position 1 to the nucleotide in position 792 of the sequence SEQ ID No. 62.
  • the potential coding sequence begins at the nucleotide at position 49 and ends at the nucleotide at position 792 of the sequence SEQ ID No. 62.
  • the translation initiation codon begins at the nucleotide at position 49 of the sequence SEQ ID No. 62.
  • This sequence comprises a Kozak motif of sequence "GC CGC GCC ATG C" which begins at the nucleotide in position 41 of the sequence SEQ ID No. 62.
  • transcripts detected with the sequence probe SEQ ID No. 101 are 1.9 kb in the pancreas, the kidney, the skeletal muscle, the lung and the placenta.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction in the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL.
  • the open partial reading frame of the nucleic acid sequence SEQ ID No. 62 potentially codes for a polypeptide of 263 amino acids in length constituting the sequence SEQ ID No. 158.
  • GBAS guanine nucletide-binding protein, alpha- subunit (adenylate cyclase-stimulating G alpha protein). This protein is described with a phosphorylation site and a transmembrane region.
  • the "guanine nucletide-binding proteins” have a role of modulators or transducers in different transmembrane signaling systems.
  • the polypeptide of sequence SEQ ID No. 158 includes glycosylation sites, phosphorylation sites such as cAMP- and cGMP-dependent protein kinase and casein kinese type II phosphorylation sites.
  • polypeptide of sequence SEQ ID No. 158 is therefore related by its motif found in the domain PD013981 of PRODOM: (homologous domains detected in Swissprot, version 34.2, November 1997) to a protein putatively involved in vesicular intracellular transport. This mechanism, underpinned by a specific addressing mediated by these proteins, is of interest compared to Tangier disease / FHD which is described by a defect in the mechanism of transport and tra ⁇ slocation of pools of intracellular cholesterol towards the accepting HDL particles. The polypeptide of sequence SEQ ID No. 158 is therefore likely to intervene in an important stage involved in the reverse transport of cholesterol by HDL.
  • polypeptide of sequence SEQ ID No. 158 is capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier disease, in family deficiencies in HDL, or in a disease genetically linked to the locus 9q31-34 of chromosome 9.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS98601 has been isolated according to the invention. Three nucleic acid sequences representative of this transcript have been determined. The first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 63.
  • sequence SEQ ID No. 63 is 335 nucleotides in length.
  • the second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 64.
  • sequence SEQ ID No. 64 is 447 nucleotides in length. No sequence identity was found during a search in the GenBank database (Version 110).
  • the third nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 65.
  • sequence SEQ ID No. 65 is 2324 nucleotides in length. This sequence includes a partial open reading frame (ORF) going from the nucleotide in position 3 to the nucleotide in position 611 of the sequence SEQ ID No. 65.
  • ORF partial open reading frame
  • the analyzes of expression of the transcript of sequence SEQ ID No. 45 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS98601 gene was expressed in the brain, placenta and uterus.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • the open reading frame of the third nucleic acid sequence SEQ ID No. 65 potentially codes for a polypeptide of 203 amino acids in length constituting the sequence SEQ ID No. 159. Homologies observed at the level of the protein sequence (307):
  • polypeptide of sequence SEQ ID No. 159 is capable of intervening in the regulation of the flow of cholesterol, and more particularly of Tangier's disease, in family deficiencies in HDL, or in a disease genetically linked to the locus 9q31-34 of chromosome 9.
  • This gene is a candidate by its chromosomal location for Tangier / FHD pathologies.
  • Nucleic acid A messenger RNA corresponding to a transcript of the gene designated here under the number GS94852 has been isolated according to the invention. Three nucleic acid sequences representative of this transcript have been determined.
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 66.
  • sequence SEQ ID No. 66 is 447 nucleotides in length.
  • the second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 67.
  • SEQ ID NO: 67 is 564 nucleotides in length.
  • sequence SEQ ID No. 66 two nucleotide primers were synthesized, the primers of sequences SEQ ID No. 115 and 116 respectively. From the sequence SEQ ID No. 67, two nucleotide primers were synthesized, the primers of sequences SEQ ID N ° 1 17 and 1 18 respectively.
  • the primers of sequences SEQ ID N ° 115 to 118 made it possible to amplify a cDNA, from a bank of polyA + mRNA of different human tissues marketed by the Company Clontech, a third nucleic sequence of the cDNA corresponding to the transcript of the gene
  • the third nucleic sequence corresponding to the transcript of the GS94852 gene constitutes the sequence SEQ ID No. 68.
  • the sequence SEQ ID No. 68 has a length of 604 nucleotides.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or to a disease genetically linked to locus 9q31 -34 of the chromosome 9.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS935135 has been isolated according to the invention. Three nucleic acid sequences representative of this transcript have been determined.
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 69.
  • sequence SEQ ID No. 69 is 482 nucleotides in length.
  • the second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 70.
  • sequence SEQ ID No. 70 is 402 nucleotides in length.
  • SEQ ID No 119 from the sequence SEQ ID No 69 and a second nucleotide primer of sequence SEQ ID N 120 from the sequence SEQ ID No. 70. These primers made it possible to amplify a third nucleic sequence representative of the transcript of the gene GS935135 constituting the sequence SEQ ID No. 71.
  • the nucleic sequence SEQ ID NO: 71 is 758 nucleotides in length.
  • the expression analyzes of the transcript of sequence SEQ ID No. 69 or 70 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS935135 gene was expressed in the fetal brain, the liver, the brain, the prostate, the placenta, the fetal liver, the uterus, the testicle. and the kidney. This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or to a disease genetically linked to locus 9q31 -34 of the chromosome 9.
  • Nucleic acid (s) II was isolated according to the invention a messenger RNA corresponding to a transcript of the gene designated here under the number GS914669. Three nucleic acid sequences representative of this transcript have been determined.
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 72.
  • sequence SEQ ID No. 72 is 673 nucleotides in length.
  • the second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 73.
  • the sequence SEQ ID No. 73 is 554 nucleotides in length.
  • sequence SEQ ID No. 73 From the sequence SEQ ID No. 73, two nucleotide primers were synthesized, the primers of sequences SEQ ID No. 123 and 124 respectively.
  • the primers of sequences SEQ ID N ° 121 to 124 made it possible to amplify a cDNA, from a polyA + mRNA library of different human tissues marketed by the Clontech Company, a third nucleic sequence of the cDNA corresponding to the transcript of the GS914669 gene. This sequence constitutes the sequence SEQ ID No. 74.
  • the sequence SEQ ID No. 74 has a length of 1794 nucleotides.
  • This sequence includes an open reading frame going from the nucleotide in position 1 to the nucleotide in position 258 of the sequence SEQ ID No. 74 as well as an identically localized coding sequence.
  • This sequence includes a polyadenylation site starting at the nucleotide at position 1751 of the
  • the expression analyzes of the transcript of sequence SEQ ID No. 72 or 73 were carried out by RT PCR, as described in Example 1.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or deficiencies. in HDL, or to a disease genetically linked to locus 9q31-34 on chromosome 9.
  • Polypeptide encoded by the nucleic acid of sequence SEQ ID No. 74 The open reading frame of the nucleic acid sequence SEQ
  • Nucleic acid (s) II was isolated according to the invention a messenger RNA corresponding to a transcript of the gene designated here under the number GS913839. Three nucleic acid sequences representative of this transcript have been determined.
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 75.
  • sequence SEQ ID No. 75 is 507 nucleotides in length.
  • the second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 76.
  • the sequence SEQ ID No. 76 is 415 nucleotides in length.
  • sequence identity with the sequences SEQ ID N ° 75 and 76 was found during a search in the GenBank database (Version 1 10). From the sequence SEQ ID No. 75, a nucleotide primer of sequence SEQ ID No. 125 was synthesized.
  • sequence SEQ ID No. 76 a nucleotide primer of sequence SEQ ID No. 1 126 was synthesized.
  • the primers of sequences SEQ ID No. 125 and 126 made it possible to amplify a cDNA, from a polyA + mRNA bank of different human tissues marketed by the Clontech Company a third nucleic sequence of the cDNA corresponding to the transcript of the GS94852 gene. This sequence constitutes the sequence SEQ ID No. 77.
  • the sequence SEQ ID No. 77 is 1318 nucleotides in length.
  • the expression analyzes of the transcript of sequence SEQ ID No. 75 or 76 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues have demonstrated that the GS913839 gene was expressed in the fetal brain and liver.
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS912639 has been isolated according to the invention. Three nucleic acid sequences representative of this transcript have been determined.
  • the first nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 78.
  • sequence SEQ ID No. 78 is 530 nucleotides in length.
  • the second nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 79.
  • sequence SEQ ID No. 79 is 495 nucleotides in length.
  • nucleotide primer of sequence SEQ ID No. 127 was synthesized.
  • sequence SEQ ID No. 79 a nucleotide primer of sequence SEQ ID No. 128 was synthesized
  • the primers of sequences SEQ ID N ° 127 and 128 made it possible to amplify a cDNA, from a bank of polyA + mRNA of different human tissues marketed by the Clontech Company, a third nucleic sequence of the cDNA corresponding to the transcript of the GS912639 gene. This sequence constitutes the sequence SEQ ID N ° 80.
  • sequence SEQ ID No. 80 is 594 nucleotides in length.
  • the analyzes of expression of the transcript of sequence SEQ ID No. 78 or 79 were carried out by RT PCR, as described in Example 1. These analyzes carried out using polyA + RNA from different tissues made it possible to show that the GS912639 gene was expressed in the liver. This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • a messenger RNA corresponding to a transcript of the gene designated here under the number GS933630 has been isolated according to the invention.
  • a nucleic acid sequence representative of this transcript has been determined.
  • This nucleic sequence of the cDNA corresponding to this transcript constitutes the sequence SEQ ID No. 81.
  • sequence SEQ ID No. 81 has a length of 582 nucleotides. No homology was observed with the sequences referenced in the GenBank database (Version 116).
  • This gene constitutes a causal positional candidate for a disease due to a dysfunction of the reverse flow of cholesterol, and more particularly Tangier's disease or family deficiencies in HDL, or a disease genetically linked to locus 9q31-34 of the chromosome 9.
  • the invention thus relates to a nucleic acid coding for a protein having an amino acid sequence chosen from the group of amino acid sequences SEQ ID No. 129 to SEQ ID No. 160 or a peptide fragment or a variant thereof or a nucleic acid of complementary sequence.
  • the nucleic acids according to the invention are in an isolated or purified form.
  • the invention also relates to a nucleic acid comprising at least eight consecutive nucleotides of a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID No. 1 to SEQ ID No. 81 and SEQ ID No. 82 to SEQ ID No. 101, or a nucleic acid of complementary sequence.
  • the invention also relates to a nucleic acid comprising at least 20, 30, 40, 50, 100 or 150 consecutive nucleotides of a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID No. 1 to SEQ ID No. 81 and SEQ ID No. 82 to SEQ ID No. 101, or a nucleic acid of complementary sequence.
  • the invention also relates to a nucleic acid having at least 90% nucleotide identity with a nucleic acid chosen from the group consisting of the nucleotide sequences SEQ ID No. 1 to SEQ ID No. 81 and SEQ ID N ° 82 to SEQ ID No. 101, advantageously 80%, preferably 95, 99%, 99.5%, and very preferably 99.8% of nucleotide identity with a nucleic acid chosen from the group consisting of nucleotide sequences SEQ ID No. 1 to SEQ ID No. 81 and SEQ ID No. 82 to SEQ ID No. 101, or a nucleic acid of complementary sequence.
  • the invention relates to a nucleic acid hybridizing, under hybridization conditions of high stringency, with a nucleic acid as defined above, and more particularly a nucleic acid chosen from the group consisting of nucleotide sequences SEQ ID No. 1 to SEQ ID No. 81 and SEQ ID No. 82 to SEQ ID No. 101, or a nucleic acid of complementary sequence.
  • each of the nucleotide sequences SEQ ID No. 1 to SEQ ID No. 81 constitute cDNAs whose nucleotide sequence is found in the transcripts of genes potentially involved in disorders linked to a dysfunction of metabolism lipoproteins, in particular the reverse transport of cholesterol.
  • an open reading phase has been determined, which codes for a polypeptide whose alteration in the amino acid sequence or in the expression is potentially associated with one of these conditions, which indicates that the nucleotide sequences comprising the open reading phases constitute nucleic acids of potentially therapeutic interest.
  • the subject of the invention is also a nucleic acid having at least 80% nucleotide identity with a polynucleotide comprising, or alternatively consisting of, a complete or partial open reading frame, as defined above in this description.
  • nucleic acids which comprise, totally or partially, the coding region of the transcripts of sequences SEQ ID No. 1 to SEQ ID No. 81 can be expressed in a desired host cell, when these nucleic acids are placed under the control of suitable expression signals.
  • Such expression signals can be either the expression signals contained in the regulatory regions of each of the corresponding genes or, on the contrary, made up of exogenous regulatory nucleic sequences.
  • nucleic acid placed under the control of a functional regulatory sequence in the desired host cell can also be inserted into a vector for expression.
  • nucleic acid fragments derived from any of the nucleotide sequences SEQ ID No. 1 to SEQ ID No. 81 are useful for detecting the presence of at least one copy of a nucleotide sequence chosen from the sequences SEQ ID N ° 1 to SEQ ID N ° 81 or a fragment or a variant thereof in a sample.
  • the nucleotide probes or primers according to the invention comprise at least eight consecutive nucleotides of a nucleic acid chosen from the group consisting of sequences SEQ ID No. 1 to SEQ ID No. 81, or of a nucleic acid of complementary sequence.
  • nucleotide probes or primers according to the invention will have a length of 10, 12, 15, 18 or 20 to 25, 35, 40, 50, 70, 80, 100, 200, 500, 1000, 1500 consecutive nucleotides d a nucleic acid according to the invention, in particular a nucleic acid of nucleotide sequence chosen from the sequences SEQ ID No. 1 to SEQ ID No. 81 or of a nucleic acid of complementary sequence.
  • a probe or a nucleotide primer according to the invention will consist and / or include the fragments of a length of 12, 15, 18, 20, 25, 35, 40, 50, 100, 200, 500, 1000, 1500 nucleotides consecutive of a nucleic acid according to the invention, more particularly of a nucleic acid chosen from the sequences SEQ ID No. 1 to SEQ ID No. 81, or of a nucleic acid of complementary sequence.
  • the definition of a probe and of a nucleotide primer according to the invention therefore includes oligonucleotides which hybridize, under the conditions of high stringency hybridization defined above, with a nucleic acid chosen from the sequences SEQ ID No. 1 to SEQ ID No.
  • probes and primers according to the invention comprise all or part of a polynucleotide chosen from the nucleotide sequences SEQ ID No 82 to 101, or nucleic acids of complementary sequence, or also from the nucleotide sequences SEQ ID No 102 to 128, or nucleic acids of complementary sequence.
  • a primer or a nucleotide probe according to the invention can be prepared by any suitable method well known to those skilled in the art, including by cloning and action of restriction enzymes or also by direct chemical synthesis according to techniques such as the method to the phosphodiester of NARANG et al. (1979) or BROWN et al. (1979), the diethylphosphoramidite method of BEAUCAGE et al. (1980) or the solid support technique described in EU Patent No. EP 0 707 592.
  • Each of the nucleic acids according to the invention can be labeled, if desired, by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical or even chemical means.
  • markers can consist of radioactive isotopes ( 32 P, 33 P,, 3 H, 35 S,), fluorescent molecules (5-bromodeoxyuridine, fluorescein, acetylaminofluorene, digoxigenin) or also ligands such as biotin .
  • the labeling of the probes is preferably done by incorporating labeled molecules within the polynucleotides by extension of primers, or else by adding to the 5 ′ or 3 ′ ends.
  • the probes according to the invention can have structural characteristics such as to allow amplification of the signal, such as the probes described by URDEA et al. (1991) or in European patent n ° EP-0 225 807 (CHIRON).
  • oligonucleotide probes according to the invention can be used in particular in Southern type hybridizations with genomic DNA or also in hybridizations with the corresponding messenger RNA when the expression of the corresponding transcript is sought in a sample.
  • the probes according to the invention can also be used for the detection of PCR amplification products or even for the detection of mismatches.
  • Nucleotide probes or primers according to the invention can be immobilized on a solid support.
  • solid supports are well known to those skilled in the art and include surfaces of the wells of microtiter plates, polystyrene beds, magnetic beds, nitrocellulose strips, or even microparticles such as latex particles.
  • the present invention also relates to a method for detecting the presence of a nucleic acid as described above in a sample, said method comprising the steps of: 1) bringing one or more nucleotide probes according to the invention into contact with the sample to be tested;
  • the oligonucleotide probe (s) are immobilized on a support.
  • the oligonucleotide probes include a detectable marker.
  • the invention further relates to a kit or kit for detecting the presence of a nucleic acid according to the invention in a sample, said kit comprising: a) one or more nucleotide probes as described above; b) where appropriate, the reagents necessary for the hybridization reaction.
  • the detection kit or kit is characterized in that the probe or probes are immobilized on a support.
  • the detection kit or kit is characterized in that the oligonucleotide probes comprise a detectable marker.
  • such a kit will comprise a plurality of oligonucleotide probes in accordance with the invention which can be used to detect target sequences of interest or alternatively to detect mutations in the coding regions or the non-coding regions of the nucleic acids according to the invention, more particularly nucleic acids of sequences SEQ ID No. 1 to SEQ ID No. 81 or the nucleic acids of complementary sequence.
  • Preferred probes would include all or part of the polynucleotides of sequences SEQ ID No. 82 to SEQ ID No. 101.
  • the probes according to the invention immobilized on a support can be ordered in matrices such as "DNA chips".
  • matrices such as "DNA chips”.
  • the nucleotide primers according to the invention can be used to amplify any of the nucleic acids according to the invention, and more particularly all or part of a nucleic acid of sequences SEQ ID No. 1 to SEQ ID No. 81, or again a variant of it.
  • Another subject of the invention relates to a method for the amplification of a nucleic acid according to the invention, and more particularly a nucleic acid of sequences SEQ ID No. 1 to SEQ ID No. 81 or a fragment or a variant of this contained in a sample, said method comprising the steps of: a) bringing the sample in which the presence of the target nucleic acid is suspected with a pair of nucleotide primers whose hybridization position is localized respectively on the 5 ′ side and on the 3 ′ side of the region of the target nucleic acid whose amplification is sought, in the presence of the reagents necessary for the amplification reaction; and b) detection of the amplified nucleic acids.
  • the subject of the invention is also a kit or kit for the amplification of a nucleic acid according to the invention, and more particularly all or part of a nucleic acid of sequences SEQ ID No. 1 to SEQ ID No. 81 , said kit or kit comprising: a) a pair of nucleotide primers in accordance with the invention, the hybridization position of which is located respectively on the 5 ′ side and on the 3 ′ side of the target nucleic acid, the amplification of which is sought; b) where appropriate, the reagents necessary for the amplification reaction.
  • Such an amplification kit or kit will advantageously comprise at least one pair of nucleotide primers as described above.
  • the invention also relates to a recombinant vector comprising a nucleic acid according to the invention.
  • such a recombinant vector will comprise a nucleic acid chosen from the following nucleic acids: a) a nucleic acid coding for a protein having an amino acid sequence chosen from the group of sequences SEQ ID No. 129 to SEQ ID No. 160 or a peptide fragment or a variant thereof; b) a nucleic acid comprising a polynucleotide chosen from the group consisting of sequences SEQ ID No. 1 to SEQ ID No. 81, or a fragment or a variant thereof; c) a nucleic acid having at least 80% nucleotide identity with a nucleic acid chosen from the group consisting of sequences SEQ ID No. 1 to SEQ ID No.
  • nucleic acid hybridizing under high stringency hybridization conditions, with a nucleic acid of sequences SEQ ID No. 1 to SEQ ID No. 81, or a fragment or a variant thereof.
  • vector within the meaning of the present invention is meant a circular or linear DNA or RNA molecule which is either in the form of single strand or double strand.
  • a recombinant vector according to the invention is used in order to amplify the nucleic acid which is inserted therein after transformation or transfection of the desired cellular host.
  • they are expression vectors comprising, in addition to a nucleic acid conforming to the invention, regulatory sequences for directing transcription and / or translation.
  • a recombinant vector according to the invention will comprise in particular the following elements: (1) elements for regulating the expression of the nucleic acid to be inserted, such as promoters and enhancers;
  • the recombinant vectors according to the invention may include one or more origins of replication in cellular hosts in which their amplification or expression is sought, markers or selection markers.
  • the bacterial promoters could be the Lacl, LacZ promoters, the RNA polymerase promoters of bacteriophage T3 or T7, the PR or PL promoters of phage lambda.
  • Promoters for eukaryotic cells will include the HSV virus thymidine kinase promoter or the mouse metallothionein-L promoter.
  • the preferred bacterial vectors according to the invention are for example the vectors pBR322 (ATCC37017) or alternatively vectors such as pAA223-3 (Pharmacia, Uppsala, Sweden), and pGEMI (Promega Biotech, Madison, Wl, USA).
  • a recombinant vector according to the invention can also be a retroviral vector or also an adeno-associated vector (AAV).
  • AAV adeno-associated vector
  • Such adeno-associated vectors are for example described by FLOTTE et al. (1992), SAMULSKI et al. (1989), or even McLAUGHLIN BA et al.
  • the invention also relates to a recombinant host cell comprising a nucleic acid according to the invention, and more particularly a nucleic acid of sequences SEQ ID No. 1 to SEQ ID No. 81 or also a nucleic acid comprising all or part of the region coding of these.
  • the invention also relates to a recombinant host cell comprising a recombinant vector as described above.
  • the preferred host cells according to the invention are for example the following:
  • prokaryotic host cells Escherichia coli strains (strain DH5- ⁇ ), Bacillus subtilis, Salmonella typhimurium, or strains of species such as Pseudomonas, Streptomyces and Staphylococus;
  • the invention relates to a polypeptide comprising an amino acid sequence chosen from the group consisting of peptides of sequences SEQ ID No. 129 to SEQ ID No. 160, or a peptide fragment or a variant thereof.
  • the invention also relates to a polypeptide comprising at least
  • the invention also relates to a polypeptide comprising an amino acid sequence having at least 80% amino acid identity with an amino acid sequence chosen from the group consisting of peptides of sequences SEQ ID No. 129 to SEQ ID
  • No. 160 or a peptide fragment or a variant thereof.
  • part of the invention is a polypeptide having at least 85%, 90%, 95% or 99% identity in amino acids with an amino acid sequence chosen from the group consisting of peptides of sequences SEQ ID No. 129 to SEQ ID No. 160, or a peptide fragment or a variant thereof.
  • polypeptides according to the invention will have a length of 15, 18 or 20 to 25, 35, 40, 50, 70, 80, 100 or 200 consecutive amino acids of a nucleic acid according to the invention, in particular a amino acid sequence polypeptide chosen from the sequences SEQ ID No. 129 to SEQ ID No. 160.
  • a polypeptide according to the invention will consist and / or include fragments with a length of 15, 18, 20, 25, 35, 40, 50, 100 or 200 consecutive amino acids of a polypeptide according to the invention, more particularly a polypeptide chosen from the sequences SEQ ID No. 129 to SEQ ID No. 160.
  • polypeptides according to the present invention are in an isolated or purified form.
  • the invention also relates to a polypeptide comprising amino acid modifications of 1, 2, 3, 4, 5, 10 to 20 substitutions, additions or deletions of an amino acid with respect to the amino acid sequence of a polypeptide of sequences SEQ ID No. 129 to SEQ ID No. 160, or of a fragment or a variant thereof.
  • the invention also relates to a process for the production of one of the polypeptides of sequences SEQ ID No. 129 to SEQ ID No. 160 or of a peptide fragment or a variant thereof, said method comprising the following: steps of: a) inserting a nucleic acid encoding said polypeptide into an appropriate vector; b) cultivating, in an appropriate culture medium, a host cell previously transformed or transfected with the recombinant vector of step a); c) recovering the conditioned culture medium or lysing the host cell, for example by sonication or by osmotic shock; d) separating and purifying from said culture medium or also from the cell lysates obtained in step c), said polypeptide; e) where appropriate, characterize the recombinant polypeptide produced.
  • the peptides according to the invention can be characterized by attachment to an immunoaffinity chromatography column on which the antibodies directed against this polypeptide or against a fragment or a variant of the latter have been immobilized beforehand.
  • a recombinant polypeptide according to the invention can be purified by passage through an appropriate series of chromatography columns, according to the methods known to those skilled in the art and described for example in F. Ausubel et al (1999 ).
  • a polypeptide according to the invention can also be prepared by conventional techniques of chemical synthesis either in homogeneous solution or solid phase.
  • a polypeptide according to the invention may be prepared by the technique or in a homogeneous solution described by HOUBENWEYL (1974) or also the solid phase synthesis technique described by MERRIFIELD (1965a; 1965b). Also part of the invention are polypeptides called "homologous" to any of the polypeptides of amino acid sequences SEQ ID No. 129 to SEQ ID No. 160, or their fragments or variants. Such homologous polypeptides have amino acid sequences having one or more substitutions of an amino acid with an equivalent amino acid, relative to the reference polypeptides.
  • the equivalent amino acid according to the present invention will be understood, for example replacement of a residue in the L form with a residue in the D form or alternatively the replacement of a glutamic acid (E) by a pyro-glutamic acid according to techniques well known to those skilled in the art.
  • E glutamic acid
  • a pyro-glutamic acid a pyro-glutamic acid according to techniques well known to those skilled in the art.
  • the synthesis of peptide containing at least one residue in the D form is described by KOCH (1977).
  • two amino acids belonging to the same class are also considered to be equivalent amino acids, that is to say two amino acids, basic, non-polar or even uncharged polar.
  • polypeptides comprising at least one non-peptide bond such as a retro-inverso bond (NHCO), a carba bond (CH 2 CH 2 ) or even a ketomethylene bond (CO-CH 2 ).
  • NHCO retro-inverso bond
  • CH 2 CH 2 carba bond
  • CO-CH 2 ketomethylene bond
  • polypeptides according to the invention comprising one or more additions, deletions, substitutions of at least one amino acid will retain their capacity to be recognized by antibodies directed against the unmodified polypeptides.
  • polypeptides according to the invention in particular the polypeptides of amino acid sequences SEQ ID No 129 to SEQ ID No 160 or the fragments and variants thereof as well as the homologous peptides can be used for the preparation of antibodies .
  • antibody within the meaning of the present invention, is meant in particular polyclonal or monoclonal antibodies or fragments (for example fragments F (ab) ' 2 , Fab) or any polypeptide comprising a domain of the initial antibody recognizing the polypeptide or the target polypeptide fragment according to the invention.
  • Monoclonal antibodies can be prepared from hybridomas using the technique described by KOHLER and MILSTEIN (1975).
  • the present invention also relates to antibodies directed against a polypeptide as described above or a fragment or a variant thereof, as produced in the trioma technique or also the hybridoma technique described by KOZBOR et al. (1983).
  • the invention also relates to fragments of single chain Fv antibody (ScFv) as described in US Patent No. 4,946,778 or by MARTINEAU et al. (1998).
  • the antibodies according to the invention also include fragments of antibodies obtained using phage banks RIDDER et al., (1995) or even humanized antibodies REIMANN et al. (1997); LEGER et al., (1997).
  • the antibody preparations according to the invention are useful in immunological detection tests intended to identify the presence and / or the quantity of antigens present in a sample.
  • An antibody according to the invention may also comprise an detectable isotopic or non-isotopic marker, for example fluorescent or also be coupled to a molecule such as biotin, according to techniques well known to those skilled in the art.
  • the subject of the mention is furthermore a method for detecting the presence of a polypeptide in accordance with the invention in a sample, said method comprising the steps of: a) bringing the sample to be tested into contact with an antibody such as described above; b) detecting the antigen / antibody complex formed.
  • the invention also relates to a kit or kit for diagnosis or for the detection of the presence of a polypeptide according to the invention in a sample, said kit comprising: a) an antibody as defined above; b) a reagent allowing the detection of the antigen / antibody complexes formed.
  • a polypeptide according to the invention can be used to screen for molecules which bind to it.
  • the binding of the polypeptide with the molecule or substance can activate (agonist molecule) or inhibit (antagonist molecule) the activity of said polypeptide.
  • Such molecules capable of binding to any one of the polypeptides according to the invention include antibodies, oligonucleotides, other proteins and generally small molecules of all kinds.
  • a screening test for a candidate molecule capable of binding to a polypeptide according to the invention may advantageously comprise a first step during which the polypeptide of interest or the candidate molecule is immobilized on a support. , a second step during which the second partner (candidate molecule or polypeptide of interest) is placed in the presence of the first compound previously immobilized on the support, a third step during which one or more washes are carried out under appropriate conditions elimination of the compounds which are not specifically linked, and finally a fourth stage during which the any complex formed between the polypeptide of interest and the candidate molecule is detected.
  • the detection of the complex formed by the candidate molecule and the polypeptide of interest according to the invention can be advantageously carried out with using an antibody as described above.
  • the candidate molecule will advantageously be labeled with the aid of a detectable marker prior to its brought into contact with the immobilized polypeptide of interest.
  • Such a detectable marker can be radioactive or non-radioactive, for example fluorescent or correspond to a ligand for a third partner used for detection such as a biotin molecule.
  • the subject of the invention is also a process for screening a molecule or a candidate substance interacting with a polypeptide according to the invention, said method comprising the steps of: a) bringing into contact a polypeptide in accordance with invention with the candidate substance or molecule to be tested; b) detecting the complexes possibly formed between said polypeptide and said substance or candidate molecule.
  • the invention also relates to a kit or kit for screening a molecule or a candidate substance interacting with a polypeptide according to the invention, said kit comprising: a) a polypeptide according to the invention; b) if necessary, means necessary for the detection of the complex formed between said polypeptide and the candidate molecule or substance.
  • kit or kit for screening a molecule or a candidate substance interacting with a polypeptide according to the invention comprising: a) a polypeptide according to the invention; b) if necessary, means necessary for the detection of the complex formed between said polypeptide and the candidate molecule or substance.
  • the expression profile of the polynucleotides according to the present invention is determined according to the Northern blot analysis and reverse transcription coupled to PCR protocols described in particular by Sambrook et al (ref. CSH Sambrook, J., Fritsch, EF, and Maniatis, T. (1989) "Molecular Cloning: A Laboratory Manual”. 2nd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)...
  • a pair of primers synthesized from any of the nucleotide sequences of the transcripts SEQ ID No. 1 to SEQ ID No. 81 is used to detect the cDNA corresponding.
  • PCR polymerase chain reaction
  • cDNA templates corresponding to polyA + mRNAs (Clontech) retrotranscribed.
  • Reverse transcription into cDNA is carried out with the enzyme SUPERSCRIPT II (GibcoBRL, Life Technologies) according to the conditions described by the manufacturer.
  • the polymerase chain reaction is carried out according to standard conditions, in 20 ⁇ l of reaction mixture with 25 ng of the cDNA preparation.
  • the reaction mixture is composed of 400 ⁇ M of each of the dNTPs, of 2 units of Thermus aquaticus (Taq) DNA polymerase (Ampli Taq Gold; Perkin Elmer), of 0.5 ⁇ M of each primer, of 2.5 mM MgCI2, and of PCR buffer.
  • Taq Thermus aquaticus
  • PCR cycles (30 s denaturation at 94 ° C, 30 s hybridization decomposed as follows during the 34 cycles: 64 ° C 2 cycles, 61 ° C 2 cycles, 58 ° C 2 cycles and 55 ° C 28 cycles and an extension of one minute per kilobase at 72 ° C) are carried out after a first step denaturation at 94 ° C for 10 min in a Perkin Elmer 9700 thermocycler.
  • the PCR reactions are visualized on agarose gel by electrophoresis.
  • the cDNA fragments obtained can be used as probes for analysis by Northern blot and can also be used for the exact determination of the polynucleotide sequence.
  • a cDNA probe produced as described above is labeled with 32 P using the High Prime DNA labeling system (Boehringer) according to the instructions indicated by the manufacturer. After labeling, the probe is purified on a microcolumn of Sephadex G50 (Pharmacia) according to the instructions
  • the labeled and purified probe is then used for the detection of the expression of mRNAs in different tissues.
  • the Northern blot containing RNA samples from different human tissues ((Multiple Tissue Northern, MTN, Clontech) Blot 2, reference 77759-1) is hybridized with the labeled probe.
  • Example 2 Obtaining complete cDNA fragments corresponding to the transcripts according to the invention
  • a complete clone can be directly isolated by hybridization by screening a cDNA library by means of a polynucleotide probe specific for the sequence of the gene of interest.
  • a specific probe of 30-40 nucleotides is synthesized using a Applied Biosystem / Perkin Elmer brand synthesizer according to the chosen sequence.
  • the oligonucleotide obtained is radiolabelled, for example with 32 P- ⁇ -ATP using T4 polynucleotide kinase and is purified according to the usual methods (eg Maniatis et al. Molecular cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, NY 1982 or F.Ausubel et al. (Current Protocols in Molecular Biology, J. Wiley and Sons Eds, 1999).
  • the bank of clones containing the cDNA that we want to screen is spread on culture medium in a Petri dish (1.5% agar) containing the appropriate antibiotics according to the usual methods mentioned above (F. Ausubel et al.).
  • the colonies thus produced after incubation are transferred to nitrocellulose filters and screened using the radiolabelled nucleotide probe, according to the usual methods and the colonies hybridizing with the probe are isolated and subcloned.
  • the DNA of the clones thus identified is prepared and analyzed by sequencing.
  • the clones containing the fragments corresponding to the complete cDNA are purified and recloned in the vector pcDNA3 according to the protocols known to those skilled in the art and presented for example in F. Ausubel et al (1999).
  • RNA oligonucleotide is ligated to the 5 'end of an mRNA population.
  • a set of primers specific respectively to the adapter ligated in 5 ′ and of a sequence located in 3 ′ of the gene of interest is used in PCR to amplify the 5 ′ portion of the sought cDNA.
  • the amplified fragment is then used to reconstruct the complete cDNA.
  • Verification of the loss of expression of the candidate gene causing the Tangier cell phenotype can be determined by hydridation of these sequences with probes corresponding to MRNA from fibroblasts from subjects with or without the disease, according to the methods described below:
  • Total RNAs are obtained from cell cultures of fibroblasts from normal subjects or those suffering from Tangier disease by the guanidine isothiocyanate method (Chomczynski & Sacchi, 1987).
  • the poly (A) + mRNAs are obtained by affinity chromatography on oligo (dT) -cellulose columns (Sambrook et al., 1989) and the cDNAs used as probes are obtained by RT-PCR (DeRisi et al., 1997) with oligonucleotides labeled with a fluorescent product (Amersham Pharmacia Biotech; CyDye TM).
  • the glass membranes containing the sequences presented in this patent application, corresponding to the Tangier gene, are hydrided with the cDNA probes, obtained from fibroblasts (lyer et al., 1999).
  • the use of the Amersham / molecular Dynamics system (Avalanche Microscanner TM) allows the quantification of the expressions of the sequence products on the healthy or affected cell type.
  • a typical eukaryotic expression vector contains a promoter which allows the initiation of transcription of mRNA, a protein coding sequence and signals required for transcription termination and polyadenylation of the transcript. It also contains additional signals such as enhancers, the (de) Kozak sequence and sequences necessary for splicing the mRNA.
  • An efficient transcription is obtained with the early and late elements of the promoters of the SV40 virus, the retroviral LTRs or the early promoter of the CMV virus.
  • cellular elements such as the actin promoter can also be used.
  • Many expression vectors can be used to implement the present invention such as the vector pcDNA3.
  • polypeptide corresponding to the partial transcript of the GS gene No. XX or to the full cDNA described in Example 2 can be easily produced in a bacterial expression system, of insect cells using the baculovirus vectors or in mammalian cells with or without the vaccinia virus vectors. All the methods are today widely described and known to those skilled in the art. A detailed description can be found, for example, in F. Ausubel et al. (1999).
  • Example 6 Production of an antibody derived from a polypeptide
  • the antibodies in the present invention can be prepared by various methods (Current Protocols In Molecular Biology Volume 1 edited by Frederick M. Ausubel, Roger Brent, Robert E. Scientific, David D. Moore, JG Seidman, John A. Smith, Kevin Struhl - Massachusetts General Hospital Harvard Medical School, chapter 11).
  • cells expressing a polypeptide of the present invention are injected into an animal to induce the production of serum containing the antibodies.
  • the proteins are prepared and purified in order to avoid contamination. Such a preparation is then introduced into the animal in order to produce polyclonal antisera of greater activity.
  • the antibodies of the present invention are monoclonal antibodies.
  • Such monoclonal antibodies can be prepared using the hybridoma technique. (K ⁇ hler et al, Nature 256: 495 (1975); Kôhler et al, Eur. J. Immunol. 6: 51 1 (1976); Kôhler et al, Eur. J. Immunol. 6: 292 (1976); Hammeling and al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, NY, pp. 563- 681 51981).
  • such methods involve immunizing the animal (preferably a mouse) with a polypeptide or, better still, with a cell expressing the polypeptide.
  • These cells can be cultured in a suitable tissue culture medium. However, it is preferable to culture the cells in an Eagle medium (modified Earle) supplemented with 10% fetal bovine serum (inactivated at 56 ° C) and supplemented with approximately 10 g / l of non-essential amino acids, of 1000 U / ml of penicillin and approximately 100 ⁇ g / ml of streptomycin.
  • Eagle medium modified Earle
  • fetal bovine serum inactivated at 56 ° C
  • approximately 10 g / l of non-essential amino acids 1000 U / ml of penicillin and approximately 100 ⁇ g / ml of streptomycin.
  • the splenocytes of these mice are extracted and fused with an appropriate myeloma cell line.
  • an appropriate myeloma cell line SP2O
  • the parental myeloma cell line SP2O
  • the resulting hybridoma cells are selectively maintained in HAT medium and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80: 225-232 (1981)).
  • the hybridoma cells obtained after such a selection are tested in order to identify the clones secreting antibodies capable of binding to the polypeptide.
  • other antibodies capable of binding to the polypeptide can be produced according to a 2-step procedure.
  • the antibodies specific for the protein are used to immunize an animal, preferably a mouse.
  • the splenocytes of this animal are then used to produce hybridoma cells, and these cells are screened to identify clones that produce an antibody whose ability to bind to the specific protein-antibody complex may be blocked by the polypeptide.
  • These antibodies can be used to immunize an animal to induce the formation of more specific antibodies to the protein.
  • Fab and F (ab ') 2 and the other antibody fragments of the present invention could be used according to the methods described herein.
  • Such fragments are typically produced by proteolytic cleavage using enzymes such as Papain (to produce the Fab fragments) or Pepsin (to produce the F (ab ') 2 fragments).
  • secret fragments recognizing the protein can be produced by applying recombinant DNA technology or synthetic chemistry.
  • chimeric "humanized" monoclonal antibodies For the in vivo use of antibodies in humans it would be preferable to use chimeric "humanized" monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known to those of skill in the art.
  • Tangier's disease is characterized by accelerated catabolism of high density lipoprotein particles (HDL) and an accumulation of cholesterol in the tissues.
  • HDL high density lipoprotein particles
  • the skin fibroblasts of patients with Tangier's disease have a reduced capacity to eliminate their cholesterol content by the cholesterol efflux process ensured by the apolipoprotein Al (apoA-1), major protein of HDL ( Francis et al., 1995).
  • apoA-1 apolipoprotein Al
  • This characteristic corresponding to a loss of function is also found in other fibroblastic cells of patients with familial HDL deficiency (Marcil and
  • fibroblast cultures of normal subjects and of subjects suffering from Tangier disease The primary fibroblasts of human skin are obtained by culturing a skin biopsy from the forearm. These biopsies are performed on patients with Tangier's disease having the clinical and biochemical characteristics of "homozygotes", ie orange tonsils, 0 plasma concentrations of apoA-1 and HDL-cholesterol lower than 5 th percentile. Normal fibroblast lines are obtained from the American Type Culture Collection (Rockville, MD).
  • the fibroblasts are cultured in an EMMEM medium (Eagle-modified minimium essential medium; GIBCO) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 IU / ml of penicillin and 100 ⁇ g / ml of steptomycin ( medium designated by EMMEM10).
  • EMMEM medium Eagle-modified minimium essential medium; GIBCO
  • GIBCO EMMEM medium
  • these cells are preloaded with cholesterol by incubation for 24 hours with 50 ⁇ g / ml of cholesterol in the medium described above without calf serum but containing 2 mg / ml of bovine albumin (BSA, fraction V).
  • BSA bovine albumin
  • the fibroblasts preloaded with confluent cholesterol on 24-well plates are incubated in EMMEM10 medium and 1 ⁇ Ci / ml of 5 1, 2-3 H-cholesterol (50 Ci / mmol; Dupont; Wilmington, DE) for 48 hours. Approximately 100,000 counts per minute are obtained per well or 1,000 counts per minute per ⁇ g of cellular protein.
  • the cells are washed three times with EMMEM / BSA medium, and incubated with this medium for 24 hours before transfecting the gene of interest and starting the efflux by adding 10 ⁇ g / ml of proteoliposome containing apoA- l in EMMEM / BSA environment.
  • proteoliposomes are prepared by sonication of phosphatidylcholine and purified human apoA-1 (Jonas, 1986). Cell transfection is carried out by the calcium phosphate precipitation technique (Sambrook et al., 1989). After the efflux period, generally 20 hours, the medium is collected, centrifuged (1000 g, 5 min), and the radioactivity determined by counting in liquid scintillation. The residual radioactivity in the cells is also determined overnight after extraction of the lipids in isopropanol. The percentage of efflux is calculated by dividing the radioactivity measured in the supernatant by the sum of the radioactivities measured, in the supernatant and the cell extract.
  • the isolation of the genomic clones corresponding to a transcript is carried out by screening the BAC library of human genomic DNA (for example that provided by Mel Simon, CalTech., Ref: Kim et al. Genomics (1996), 34: 213 -218)) by PCR with primers specific for the cDNA sequence corresponding to the sequences of the transcripts SEQ ID No. 1 to SEQ ID No. 81 according to the invention, according to the method described in Example 1.
  • Example 9 Determination of polymorphism / mutation of one of the genes corresponding to the transcripts according to the invention
  • the detection of polymorphisms and or of mutations in the sequences of the transcripts can be carried out according to different protocols.
  • the method of choice is direct sequencing.
  • the mutation detection technique by direct sequencing consists in comparing the genomic sequences of the gene corresponding to the cDNA SEQ ID No. 1 to SEQ ID No. 81 obtained from at least 8 individuals (4 individuals affected by the pathology studied and 4 unaffected individuals). Sequence divergences constitute polymorphisms. All those modifying the amino acid sequence of the wild-type protein are mutations capable of affecting the function of said protein which it is interesting to consider more particularly in the case / control association studies described in Example 8.

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