Patent Document

INTRODUCTION  
         [0001]    The present invention relates generally to the fields of genetics and medicine. The present invention more particularly discloses the identification of a human hypercholesterolemia causal gene, which can be used for the diagnosis, prevention and treatment of hypercholesterolemia, and more particularly familial hypercholesterolemia ADH, as well as for the screening of therapeutically active drugs. The invention more specifically discloses that mutations in the PCSK9 gene encoding NARC-1 cause autosomal dominant hypercholesterolemia (ADH) and represent novel targets for therapeutic intervention. The invention can be used in the diagnosis of predisposition to, detection, prevention and/or treatment of cholesterol and lipoprotein metabolism disorders, including familial hypercholesterolemia, atherogenic dyslipidemia, atherosclerosis, and more generally cardiovascular diseases (CVD).  
         BACKGROUND  
         [0002]    Atherosclerosis is a disease of the arteries responsible for coronary heart disease (CVD) that underlies most deaths in industrialized countries (Lusis, 2000). Several risk factors for CHD have now been well established: dyslipidemias, hypertension, diabetes, smoking, poor diet, inactivity and stress. The most clinically relevant and common dyslipidemias are characterized by an increase in beta-lipoproteins (VLDL and LDL particles) with hypercholesterolemia in the absence or presence of hypertriglyceridemia (Fredrickson et al, 1967). An isolated elevation of LDL cholesterol is one of the most common risk factors for CVD. Twin studies (Austin et al, 1987) and family data (Perusse, 1989; Rice et al, 1991) have shown the importance of genetic factors in the development of the disease, particularly when its complications occur early in life. Mendelian forms of hypercholesterolemia have been identified: at first the autosomal dominant form (ADH) (Khachadurian, 1964) and later the autosomal recessive form (ARH), initially described as “pseudohomozygous type II hyperlipoproteinemia” (Morganroth et al, 1967).  
           [0003]    ADH is an heterogeneous genetic disorder. Its most frequent and archetypal form is Familial Hypercholesterolemia (FH) with a frequency of 1 in 500 for heterozygotes and 1 per million for homozygotes (Goldstein et al, 1973). The disease is co-dominant with homozygotes being affected earlier and more severely than heterozygotes. FH is caused by mutations in the gene that encodes the LDL receptor (Goldstein &amp; Brown, 1978) (LDLR at 19p13.1-p13.3) (MIN 143890). It is characterized by a selective increase of LDL cholesterol levels in plasma giving rise to tendon and skin xanthomas, arcus corneae and cardiovascular deposits leading to progressive and premature atherosclerosis, CHD and mortality (occurring before 55 years). The second form of ADH is Familial Defective apo B-100 (FDB) caused by mutations in the apolipoprotein B gene (APOB at 2p23-p24), encoding the ligand of the LDL receptor (Inneraty et al, 1987) (MIN 144010). The existence of a greater level of genetic heterogeneity in ADH (Saint-Jore et al, 2000) has been reported and the implication of a third locus named HCHOLA3 (formerly FH3) has been detected and mapped at 1p34.1-p32 in a French family (Varret et al, 1999) (MIM 603776). These results were confirmed by Hunt et al. in a large Utah kindred (Hunt et al, 2000).  
           [0004]    There is a strong need of identifying genes involved in hypercholesterolemia, more particularly in ADH, in order to understand the mechanisms leading to these disorders and to develop improved diagnosis and therapeutic treatment.  
         SUMMARY OF THE INVENTION  
         [0005]    The inventors have shown that mutations in the PCSK9 gene encoding NARC-1 cause autosomal dominant hypercholesterolemia. They have demonstrated that the NARC-1 protein contributes to cholesterol homeostasis. The invention thus discloses novel targets for diagnosis and therapeutic intervention for hypercholesterolemia, more particularly ADH, CVD, lipid and lipoprotein metabolism disorders, atherogenic dyslipidemia, atherosclerosis, and cardiovascular diseases.  
           [0006]    In a first aspect, the invention concerns a PCSK9 gene or a fragment thereof comprising an alteration, said alteration reducing, modifying or abolishing the activity of NARC-1. Preferably, said alteration is a nucleotide substitution. More preferably, said nucleotide substitution leads to an amino acid change in NARC-1 protein. Preferably, said amino acid change is located at or near the catalytic site or a zymogen processing of the NARC-1 protein and decreases the catalytic activity or autocatalytic cleavage of said protein or functional domain, respectively. Alternatively, the alteration affects the splicing of NARC-1 mRNA.  
           [0007]    The invention also concerns a corresponding NARC-1 protein or a fragment thereof comprising an alteration, said alteration reducing, modifying or abolishing the activity of NARC-1. Preferably, the alteration is located at the catalytic site of the NARC-1 protein and decreases its catalytic activity or at a zymogen processing site of NARC-1 and decreases its autocatalytic cleavage. In an other preferred embodiment, the alteration is located near the catalytic site of the NARC-1 protein and decreases its catalytic activity or near the zymogen processing sites of NARC-1 and decreases its autocatalytic cleavage. An aspect of the present invention concerns a method of genotyping in a subject a polymorphism of the PCSK9 gene, preferably a polymorphism disclosed in Table 2. The invention also concerns a method of associating one or several polymorphism(s) of the PCSK9 gene, preferably one or several polymorphism(s) disclosed in Table 2 to a disease or a disorder.  
           [0008]    An other aspect of this invention relates to a method of detecting the presence of or predisposition to hypercholesterolemia, more particularly ADH, or lipid and lipoprotein metabolism disorders in a subject, the method comprising detecting in a sample from the subject the presence of an alteration in the PCSK9 gene or in the NARC-1 protein, the presence of said alteration being indicative of the presence or predisposition to hypercholesterolemia, more particularly ADH, or lipid and lipoprotein metabolism disorders. In a most preferred embodiment, said alteration reduces, modifies, or abolishes the activity of NARC-1. Optionally, the method further comprises detecting the presence of an alteration in the LDL receptor and/or the apolipoprotein B in said sample.  
           [0009]    The invention also relates to a diagnostic kit comprising primers, probes and/or antibodies for detecting in a sample from a subject the presence of an alteration in the PCSK9 gene or in the NARC-1 protein, in the NARC-1 RNA or polypeptide expression, and/or in NARC-1 activity. Optionally, said diagnostic kit further comprises reagents for detecting in a sample from a subject the presence of an alteration in the LDL receptor and/or the apolipoprotein B.  
           [0010]    A further aspect of the invention relates to the use of a functional NARC-1, preferably a wild-type NARC-1 protein or a nucleic acid encoding the same, in the manufacture of a pharmaceutical composition for treating or preventing hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders in a subject. The invention also relates to the use of a biologically active compound which modulates NARC-1 activity, in the manufacture of a pharmaceutical composition for treating or preventing hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders in a subject. The invention also relates to a method for treating or preventing hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders in a subject comprising administering to said subject a functional NARC-1, preferably a wild-type NARC-1 protein or a nucleic acid encoding the same. The invention further relates to a method for treating or preventing hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders in a subject comprising administering to said subject a biologically active compound which modulates NARC-1 activity.  
           [0011]    An additional aspect of this invention relates to methods of selecting biologically active compounds that modulate the activity of NARC-1 protein, typically of an altered NARC-1 polypeptide. The compounds are more particularly suitable for treating hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders. 
       
    
    
     LEGEND TO FIGURES  
       [0012]    [0012]FIG. 1: Family HC92 Pedigree and Genetic Analysis with Markers Spanning the 1p34.1-p32 Region  
         [0013]    Affected subjects present with a history of tendon xanthomas (HC92-II-7 and III-3), CHD, early myocardial infarction (HC92-II-2 and II-6) and stroke (HC92-II-4). The affected allele is represented by the filled bars. Age (in years) at lipid measurement, total and LDL cholesterol (in g/L; untreated values for affected members) are given.  
         [0014]    [0014]FIG. 2: Genetic Analysis of Family HC2  
         [0015]    Pedigree of the HC2 family is shown. Half-blackened symbols indicate affected members, unblackened symbols indicate unaffected members, and hatched symbols indicate members with an unknown phenotypic status. The haplotype in brackets of subject HC2-I-1 was unequivocally deduced. Selected markers spanning the 1p34.1-p32 region are displayed to the left of the pedigrees. The affected allele is represented by the filled bars. Age (in years) at lipid measurement, total and LDL cholesterol (in g/L; untreated values for affected members) are given.  
         [0016]    [0016]FIG. 3: Genetic Analysis and Mutation Detection in Families HC92 and HC60  
         [0017]    a, Results of LINKMAP analyses in the HC92 family indicating a maximum lod score for D1S2742 at θ=0. PCSK9 maps 1.2 Mb to this marker. b, Mutation in family HC92. The proband (HC92-II-7) is heterozygous for a T→A substitution in exon 2 at nucleotide 625 (S127R). c, Family pedigree and genetic analysis of family HC60. d, Sequence analysis in family HC60. The proband (HC60-II-2) is heterozygous for a T→C substitution in exon 4 at nucleotide 890 predicting a substitution at 216 of leucine for the conserved phenylalanine (F216L).  
         [0018]    [0018]FIG. 4: Mutations Study  
         [0019]    a, Segregation of the S127R mutation in part of family HC2. The T→A substitution at nucleotide 625 creates a new recognition cleavage site for restriction digestion by MnlI (represented by *). After electrophoretic migration on a 2% agarose gel, fragments of 208, 203 and 60 bp were distinguished in the normal allele, while fragments of 208, 143 and 60 bp appeared in the mutated alleles (the 203 bp normal fragment was divided in fragments of 143 and 60 bp and the two 60 bp fragments generated comigrated). The proband (HC2-II-9) and one of her children (HC2-II-10) were observed to be heterozygous for the S127R mutation (as indicated by both the 203 and 143 bp bands).  
         [0020]    b, The amino acid sequence alignment for NARC-1 shows conservation of the serine at codon 127 between human, mouse and rat. DNA sequences of the normal and mutant genes are shown above and below the amino acid sequences, respectively.  
         [0021]    c, The amino acid sequence alignment for NARC-1 shows conservation of the phenylalanine at codon 216 between human, mouse and rat. DNA sequences of the normal and mutant genes are shown above and below the amino acid sequences, respectively. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0022]    Definition  
         [0023]    The PCSK9 gene (or NARC-1 gene) encodes the NARC-1 protein or polypeptide. The NARC-1 protein is translated as a pre-protein which is autocatalytically processed into a mature NARC-1 protein. The sequence of the NARC-1 gene has been described in patent applications WO 01/57081 and WO 02/14358, and partly characterized in Seidah et al (2003). The residues of the NARC-1 catalytic site consist in Asp-186, Ser-188, His-226, Asn-317 and Ser-386. NARC-1 presents two zymogen processing sites : a first one comprising residues 78 to 82 and having a primary cleavage site located at position 82; a second one comprising residues 138 to 142 and having a putative secondary cleavage site located at position 142 . The biological function of NARC-1 and the implication of this protein in hypercholesterolemia and lipid and lipoprotein metabolism disorders were unknown.  
         [0024]    Within the context of this invention, the PCSK9 gene locus designates all PCSK9 sequences or products in a cell or organism, including PCSK9 coding sequences, PCSK9 non-coding sequences (e.g., introns, 5′ and 3′ UTR), PCSK9 regulatory sequences controlling transcription and/or translation (e.g., promoter, enhancer, terminator, etc.), as well as all corresponding expression products, such as PCSK9 RNAs (e.g. mRNA) and NARC-1 polypeptides (e.g., a pre-protein and a mature protein).  
         [0025]    The term “gene” shall be construed to include any type of coding nucleic acid, including genomic DNA, complementary DNA (cDNA), synthetic or semi-synthetic DNA, as well as any form of corresponding RNA. The term gene particularly includes recombinant nucleic acids encoding NARC-1, i.e., any non naturally occurring nucleic acid molecule created artificially, e.g., by assembling, cutting, ligating or amplifying sequences. A PCSK9 gene is typically double-stranded, although other forms may be contemplated, such as single-stranded. PCSK9 genes may be obtained from various sources and according to various techniques known in the art, such as by screening DNA libraries or by amplification from various natural sources. Recombinant nucleic acids may be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof A particular example of a PCSK9 gene comprises SEQ ID NO: 1.  
         [0026]    The indicated positions in a PCSK9 gene and a NARC-1 protein refer to the positions in the sequences of SEQ ID No 1 and SEQ ID No 2, respectively.  
         [0027]    The term “hybridize under stringent conditions” means that two nucleic acid fragments are capable of hybridization to one another under standard hybridization conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (1989) Cold Spring Harbor Laboratory Press, New York, USA. More specifically, “stringent conditions” as used herein refer to hybridization at 65° C. in a hybridization buffer consisting of 250 mmol/l sodium phosphate buffer pH 7.2, 7% (w/v) SDS, 1% (w/v) BSA, 1 mmol/l EDTA and 0.1 mg/ml single-stranded salmon sperm DNA.  
         [0028]    GENE &amp; PROTEIN  
         [0029]    The invention concerns an isolated or recombinant PCSK9 gene comprising an alteration causing hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders.  
         [0030]    The altered PCSK9 gene comprises an alteration leading to a decrease or a complete loss of NARC-1 activity, or to a new NARC-1 activity. This decrease, loss, or new activity can be due to the decrease or loss of the activity of NARC-1 enzyme, to the decrease of NARC-1 stability (either at the stage of nucleic acid or proprotein or protein), to a change of substrate specificity of NARC-1 or to the disturbance or impediment of the NARC-1 polymerization. This decrease or loss of NARC-1 activity can be due to a NARC-1 alteration leading to a decrease or loss of the pro-NARC-1 maturation, either at the first cleavage or the second one or both. The alteration can also affect the catalytic activity by modifying the catalytic site of NARC-1 or its substrate recognition site. Furthermore, the alteration can affect the splicing of the NARC-1 MRNA, leading to an alternative splicing product.  
         [0031]    The invention concerns an isolated or recombinant PCSK9 gene or fragment thereof comprising an alteration, wherein said alteration reduces, modifies or abolishes the activity of NARC-1. Preferably, said alteration is a nucleotide substitution. More preferably, said nucleotide substitution leads to an amino acid change in NARC-1 protein. Preferably, said amino acid change is located at (e.g., within) the catalytic site of the NARC-1 protein and decreases its catalytic activity, or is located at a zymogen processing site of NARC-1 and decreases its autocatalytic cleavage. In an other preferred embodiment, said amino acid change is located near the catalytic site of the NARC-1 protein and decreases its catalytic activity or near the zymogen processing sites of NARC-1 and decreases its autocatalytic cleavage. Alternatively, the alteration may affect the splicing of NARC-1 mRNA. More specifically, said alteration can be is a substitution at nucleotide 625 and/or 890 of SEQ ID No 1. More preferably, said alteration is selected from the group consisting of a T→A substitution at nucleotide 625 of SEQ ID No 1, a T→C substitution at nucleotide 890 of SEQ ID No 1 and a combination thereof. In a further embodiment, said alteration is selected from the group consisting of a substitution at nucleotides 476-478 of SEQ ID No 1, a substitution at nucleotides 482-484 of SEQ ID No 1, a substitution at nucleotides 488-490 of SEQ ID No 1, a substitution at nucleotides 485-490 of SEQ ID No 1, a substitution at nucleotides 548-553 of SEQ ID No 1, a substitution at nucleotides 479-481, 491-493 and 578-580 of SEQ ID No 1, a substitution at nucleotides 620-622 of SEQ ID No 1, a substitution at nucleotides 656-658 of SEQ ID No 1, a substitution at nucleotides 671-673 of SEQ ID No 1, a substitution at nucleotides 920-922 of SEQ ID No 1, and a substitution at nucleotides 1193-1195 of SEQ ID No 1.  
         [0032]    The invention also concerns an isolated or recombinant PCSK9 gene or fragment thereof comprising at least one alteration, wherein said alteration is selected from the group consisting of the polymorphisms listed in Table 2 and in Table 4.  
         [0033]    The invention relates to an isolated or purified NARC-1 protein or a fragment thereof comprising an alteration, wherein said alteration reduces, modifies or abolishes the activity of NARC-1. Preferably, the alteration is located at the catalytic site of the NARC-1 protein and decreases its catalytic activity or at a zymogen processing site of NARC-1 and decreases its autocatalytic cleavage. In an other preferred embodiment, the alteration is located near the catalytic site of the NARC-1 protein and decreases its catalytic activity or near the zymogen processing sites of NARC-1 and decreases its autocatalytic cleavage. More preferably, said alteration can be selected from the group consisting of a substitution of the residue Serine at position 127, a substitution of the residue Phenylalanine at position 216 and a combination thereof. Still more preferably, said alteration is selected from the group consisting of a substitution of the residue Serine at position 127 of SEQ ID No 2 by an Arginine (S127R), a substitution of the residue Phenylalanine at position 216 of SEQ ID No 2 by a Leucine (F216L) and a combination thereof. In a further embodiment, said alteration is selected from the group consisting of a substitution of the residue Tyrosine at position 78 of SEQ ID No 2, a substitution of the residue Valine at position 80 of SEQ ID No 2, a substitution of the residue Leucine at position 82 of SEQ ID No 2, a substitution of the residues Valine at positions 79, 80 and 81 of SEQ ID No 2, a substitution of the residues Alanines at positions 102 and 103 of SEQ ID No 2, a substitution of the residues Valine at position 79, Lysine at position 83, and Leucine at position 112 of SEQ ID No 2, a substitution of the residue Methionine at position 126 of SEQ ID No 2, a substitution of the residue Proline at position 138 of SEQ ID No 2, a substitution of the residue Isoleucine at position 143 of SEQ ID No 2, a substitution of the residue Histidine at position 226 of SEQ ID No 2, and a substitution of the residue Asparagine at position 317 of SEQ ID No 2. Preferably, said alteration is selected from the group consisting of a substitution of the residue Tyrosine at position 78 of SEQ ID No 2 by an Alanine (Y78A), a substitution of the residue Valine at position 80 of SEQ ID No 2 by an Alanine or a Leucine (V80A or V80L), a substitution of the residue Leucine at position 82 of SEQ ID No 2 by an Alanine, a Valine or a Proline (L82A, L82V or L82P), a substitution of the residues Valine at positions 79, 80 and 81 of SEQ ID No 2 by an Arginine, an Arginine and a Leucine, respectively (V79R, V80R and V81L), a substitution of the residues Alanines at positions 102 and 103 of SEQ ID No 2 by Arginines (A102R and A103R), a substitution of the residues Valine at position 79, Lysine at position 83, and Leucine at position 112 of SEQ ID No 2 by an Isoleucine, a Methionine, a Proline, respectively (V79I, K83M and L112P), a substitution of the residue Methionine at position 126 of SEQ ID No 2 by an Alanine (M126A), a substitution of the residue Proline at position 138 of SEQ ID No 2 by a Tyrosine (P138Y), a substitution of the residue Isoleucine at position 143 of SEQ ID No 2 by a Proline (I143P), a substitution of the residue Histidine at position 226 of SEQ ID No 2 by an Alanine (H226A), and a substitution of the residue Asparagine at position 317 of SEQ ID No 2 by an Alanine (N317A). Alternatively, said alteration can be selected from the group consisting of a substitution of the residue Arginine at position 218 of SEQ ID No 2, a substitution of the residue Arginine at position 237 of SEQ ID No 2 and a combination thereof, more preferably a substitution of the residue Arginine at position 218 of SEQ ID No 2 by a Serine (R218S) or a substitution of the residue Arginine at position 237 of SEQ ID No 2 by a Tryptophane (R237W) or a combination thereof.  
         [0034]    The invention also relates to an isolated or purified NARC-1 protein or a fragment thereof comprising an alteration, wherein said alteration is selected from the group consisting of an insertion of a residue Leucine at position 15 of SEQ ID No 2, a substitution of the residue Arginine at position 46 of SEQ ID No 2 by a Leucine (R46L), a substitution of the residue Alanine at position 53 of SEQ ID No 2 by a Valine (A53V), a substitution of the residue Isoleucine at position 474 of SEQ ID No 2 by a Valine (1474V), a substitution of the residue Glutamic acid at position 670 of SEQ ID No 2 by a Glycine (E670G) and a comination thereof. The invention also relates to an isolated or purified NARC-1 protein or a fragment thereof comprising an alteration disclosed in Table 4.  
         [0035]    The invention further relates to a recombinant nucleic acid encoding a NARC-1 protein or a fragment thereof comprising an alteration according to the present invention, a vector comprising said nucleic acid, a host cell comprising said vector or said recombinant nucleic acid, and a non-human host organism comprising said recombinant nucleic acid, said vector or said host cell.  
         [0036]    Therefore, the invention concerns an isolated or recombinant PCSK9 gene and/or NARC-1 protein comprising an alteration causing hypercholesterolemia, more particularly ADH, said alteration reducing, modifying or abolishing the activity of NARC-1. In this context, by modifying is intended a change of specificity of the NARC-1 protein. Optionally, said alteration decreases or abolishes the stability of the NARC-1 protein. Optionally, said alteration decreases or abolishes the stability of MRNA encoding NARC-1. Optionally, said alteration reduces the transcription rate of the PCSK9 gene. Optionally, said alteration decreases or abolishes the activity of the NARC-1 protein. Optionally, said alteration decreases or abolishes the specificity of NARC-1 for at least one of its natural substrates. Optionally, said alteration introduces a new specificity of NARC-1 for an unusual substrate. Said unusual substrate is preferably involved in cholesterol and/or lipoprotein metabolism. Optionally, said alteration hinders or prevents the NARC-1 polymerization. Optionally, said alteration affects the catalytic site of NARC-1. Optionally, said alteration affects substrate recognition site of NARC-1. Optionally, said alteration affects the processing of pro-NARC-1 in NARC-1. More particularly, said alteration reduces or prevents the autocatalytic cleavage at one of the two zymogen processing sites or at both zymogen processing sites. Optionally, said alteration modifies the association between the NARC-1 and its prosegment, for example by increasing or decreasing their interaction.  
         [0037]    By “decrease”, it is intended within the context of this invention that the assessed parameter is between 10% and 90% of the parameter value with a wild-type NARC-1 protein in a wild-type environment. More preferably, said assessed parameter is between 25% and 75% of the parameter value with a wild-type NARC-1 protein in a wild-type environment. By “abolish”, it is intended within the context of this invention that the assessed parameter is less than 10% of the parameter value with a wild-type NARC-1 protein in a wild-type environment. More preferably, said assessed parameter is less than 5% of the parameter value with a wild-type NARC-1 protein in a wild-type environment. Still more preferably, said assessed parameter is less than 1% of the parameter value with a wild-type NARC-1 protein in a wild-type environment.  
         [0038]    In a particular embodiment, said alteration decreases or abolishes the catalytic activity of NARC-1. Preferably, said alteration is located near the catalytic site of the NARC-1 protein. Preferably, this alteration is located near a residue of the catalytic site selected from the group consisting of Aspartic acid at position 186, Serine at position 188, Histidine at position 226, Asparagine at position 317 and Serine at position 386. More preferably, this alteration is located near the histidine in position 226. Alternatively, said alteration can be located at one or several residues of the catalytic site selected from the group consisting of Aspartic acid at position 186, Serine at position 188, Histidine at position 226, Asparagine at position 317 and Serine at position 386.  
         [0039]    In an other preferred embodiment, said alteration decreases the autocatalytically cleavage of the NARC-1 protein. Preferably, said alteration is located near the zymogen processing sites of NARC-1. Said zymogen processing sites are located at positions 78-82 and 138-142. Alternatively, said alteration can be located at one or several residues of the zymogen processing sites of NARC-1.  
         [0040]    In terms of amino acid sequence, the term “near” designates,within the context of this invention, an alteration located at less than 90 amino acids, preferably 60-30 amino acids, more preferably 20 amino acids, from one residue of the catalytic site or the zymogen processing site. It is also intended that the term “near” does not include residues that form part of the catalytic site or of the zymogen processing site, as defined in the present invention.  
         [0041]    In terms of nucleotide sequence, the term “near” indicates that the alteration is located at less than 270 nucleotides, preferably 180-90 nucleotides, more preferably 60 nucleotides, from one nucleotide comprised in a codon encoding a residue of the catalytic site or of the zymogen processing site. Such alteration preferably changes the codon, thereby changing the amino acid at that position in the protein sequence.  
         [0042]    In a particular embodiment, the invention concerns an isolated or recombinant PCSK9 gene and/or an isolated or purified NARC-1 protein comprising an alteration, said alteration being preferably located at the following positions: 1-30, 32-66, 68-77, 83-225, 227-532 and 534-692 of SEQ ID No 2.  
         [0043]    Said alteration of the PCSK9 gene can be a mutation (e.g., a nucleotide substitution), a deletion or an addition of at least one nucleotide. Preferably, said alteration is a point mutation. More preferably, said mutation is selected from the group consisting of a substitution of the nucleotide at position 625 and/or 890. More preferably, said mutation is selected from the group consisting of a T→A substitution at nucleotide 625 of SEQ ID No 1, a T→C substitution at nucleotide 890 of SEQ ID No 1 and a combination thereof. In this regard, a specific object of the invention concerns a polynucleotide sequence of SEQ ID No 1 or a polynucleotide comprising a fragment of SEQ ID No 1, said polynucleotide comprising either the nucleotide A at position 625 or the nucleotide C at position 890 or a combination thereof. An other specific object of the present invention concerns a polynucleotide sequence of SEQ ID No 3 or a polynucleotide comprising a fragment of SEQ ID No 3, said polynucleotide comprising either the nucleotide A at position 5158 or the nucleotide C at position 13539 or a combination thereof.  
         [0044]    A fragment of a PCSK9 gene designates any portion of at least about 8 consecutive nucleotides of a sequence as disclosed above, preferably at least about 15, more preferably at least about 20 nucleotides, further preferably of at least 30 nucleotides. Fragments include all possible nucleotide length between 8 and 100 nucleotides, preferably between 15 and 100, more preferably between 20 and 100. Said fragment can be useful as primer or probe for identifying an alteration of the PCSK9 gene in a sample of a subject or for genotyping a PCSK9 polymorphism, preferably a polymorphism disclosed in Table 2. Said fragment can be a reagent of a diagnostic kit.  
         [0045]    The alteration of the NARC-1 protein can be a substitution, a deletion or an addition of at least one amino acid. Preferably, said alteration is a substitution. More preferably, said substitution is selected from the group consisting of a substitution of the residue Serine at position 127 of SEQ ID No 2, a substitution of the residue Phenylalanine at position 216 of SEQ ID No 2 and a combination thereof. Still more preferably, said substitution is selected from the group consisting of a substitution of the residue Serine at position 127 of SEQ ID No 2 by an Arginine (S127R), a substitution of the residue Phenylalanine at position 216 of SEQ ID No 2 by a Leucine (F216L) and a combination thereof. In this respect, a specific object of this invention concerns a polypeptide sequence of SEQ ID No 2 or a polypeptide comprising a fragment of SEQ ID No 2, said polypeptide comprising either the residue Arginine at position 127 or the residue Leucine at position 216 or a combination thereof. The invention also concerns a polynucleotide encoding said altered NARC-1 protein.  
         [0046]    A fragment of a NARC-1 protein designates any portion of at least about 8 consecutive amino acids of a sequence as disclosed above, preferably at least about 15, more preferably at least about 20 amino acids, further preferably of at least 30 amino acids. Fragments include all possible nucleotide length between 8 and 100 amino acids, preferably between 15 and 100, more preferably between 20 and 100. Said fragment can be useful for preparing antibodies.  
         [0047]    The invention also relates to an antibody specific of a NARC-1 protein comprising an alteration according to the present invention. In a preferred embodiment, said alteration causes hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders. More preferably, the invention relates to an antibody specific of a NARC-1 protein comprising a substitution of the residue Serine at position 127 of SEQ ID No 2 by an Arginine (S127R) or a substitution of the residue Phenylalanine at position 216 of SEQ ID No 2 by a Leucine (F216L) or a combination thereof. Furthermore, the invention relates to an antibody specific of a NARC-1 protein comprising an alteration selected from the group consisting of an insertion of a residue Leucine at position 15 of SEQ ID No 2, a substitution of the residue Arginine at position 46 of SEQ ID No 2 by a Leucine (R46L), a substitution of the residue Alanine at position 53 of SEQ ID No 2 by a Valine (A53V), a substitution of the residue Isoleucine at position 474 of SEQ ID No 2 by a Valine (I474V), a substitution of the residue Glutamic acid at position 670 of SEQ ID No 2 by a Glycine (E670G) and a comination thereof. Moreover, the invention relates to an antibody specific of a NARC-1 protein comprising an alteration disclosed in Table 4, preferably selected from the group consisting of a substitution of the residue Tyrosine at position 78 of SEQ ID No 2, a substitution of the residue Valine at position 80 of SEQ ID No 2, a substitution of the residue Leucine at position 82 of SEQ ID No 2, a substitution of the residues Valine at positions 79, 80 and 81 of SEQ ID No 2, a substitution of the residues Alanines at positions 102 and 103 of SEQ ID No 2, a substitution of the residues Valine at position 79, Lysine at position 83, and Leucine at position 112 of SEQ ID No 2, a substitution of the residue Methionine at position 126 of SEQ ID No 2, a substitution of the residue Proline at position 138 of SEQ ID No 2, a substitution of the residue Isoleucine at position 143 of SEQ ID No 2, a substitution of the residue Histidine at position 226 of SEQ ID No 2, and a substitution of the residue Asparagine at position 317 of SEQ ID No 2. More preferably, said alteration is selected from the group consisting of a substitution of the residue Tyrosine at position 78 of SEQ ID No 2 by an Alanine (Y78A), a substitution of the residue Valine at position 80 of SEQ ID No 2 by an Alanine or a Leucine (V80A or V80L), a substitution of the residue Leucine at position 82 of SEQ ID No 2 by an Alanine, a Valine or a Proline (L82A, L82V or L82P), a substitution of the residues Valine at positions 79, 80 and 81 of SEQ ID No 2 by an Arginine, an Arginine and a Leucine, respectively (V79R, V80R and V81L), a substitution of the residues Alanines at positions 102 and 103 of SEQ ID No 2 by Arginines (A102R and A103R), a substitution of the residues Valine at position 79, Lysine at position 83, and Leucine at position 112 of SEQ ID No 2 by an Isoleucine, a Methionine, a Proline, respectively (V791, K83M and L112P), a substitution of the residue Methionine at position 126 of SEQ ID No 2 by an Alanine (M126A), a substitution of the residue Proline at position 138 of SEQ ID No 2 by a Tyrosine (P138Y), a substitution of the residue Isoleucine at position 143 of SEQ ID No 2 by a Proline (I143P), a substitution of the residue Histidine at position 226 of SEQ ID No 2 by an Alanine (H226A), and a substitution of the residue Asparagine at position 317 of SEQ ID No 2 by an Alanine (N317A). By “specific” is intended binds specifically the altered polypeptide and essentially does not bind specifically the wild-type polypeptide or the binding of the two forms can be discriminated.  
         [0048]    Another object of the present invention is an altered PCSK9 gene having at least one nucleotide mutation at a position listed in Table 2. More particularly, the invention concerns an altered PCSK9 gene having the Leucine stretch modification, the corresponding encoded NARC-1 protein and the use thereof.  
         [0049]    A further aspect of this invention resides in novel products for use in diagnosis, therapy or screening of hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders. These products comprise nucleic acid molecules encoding a NARC-1 polypeptide according to the present invention, vectors comprising the same, recombinant host cells and expressed polypeptides.  
         [0050]    A further object of this invention is a vector comprising a nucleic acid encoding a NARC-1 polypeptide comprising an alteration according to the present invention. The vector may be a cloning vector or, more preferably, an expression vector, i.e., a vector comprising regulatory sequences causing expression of a NARC-1 polypeptide from said vector in a competent host cell.  
         [0051]    These vectors can be used to express a NARC-1 polypeptide according to the present invention in vitro, ex vivo or in vivo, to create transgenic or “Knock Out” non-human animals, to amplify the nucleic acids, to express antisense RNAs, etc.  
         [0052]    The vectors of this invention typically comprise a NARC-1 coding sequence according to the present invention operably linked to regulatory sequences, e.g., a promoter, a polyA, etc. The term “operably linked” indicates that the coding and regulatory sequences are functionally associated so that the regulatory sequences cause expression (e.g., transcription) of the coding sequences. The vectors may further comprise one or several origins of replication and/or selectable markers. The promoter region may be homologous or heterologous with respect to the coding sequence, and provide for ubiquitous, constitutive, regulated and/or tissue specific expression, in any appropriate host cell, including for in vivo use. Examples of promoters include bacterial promoters (T7, pTAC, Trp promoter, etc.), viral promoters (LTR, TK, CMV-IE, etc.), mammalian gene promoters (albumin, PGK, etc), and the like.  
         [0053]    The vector may be a plasmid, a virus, a cosmid, a phage, a BAC, a YAC, etc. Plasmid vectors may be prepared from commercially available vectors such as pBluescript, pUC, pBR, etc. Viral vectors may be produced from baculoviruses, retroviruses, adenoviruses, AAVs, etc., according to recombinant DNA techniques known in the art.  
         [0054]    In this regard, a particular object of this invention resides in a recombinant virus encoding an altered NARC-1 polypeptide according to the present invention. The recombinant virus is preferably replication-defective, even more preferably selected from E1- and/or E4-defective adenoviruses, Gag-, pol- and/or env-defective retroviruses and Rep- and/or Cap-defective AAVs. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in W095/14785, W096/22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056 and W094/19478.  
         [0055]    A further object of the present invention resides in a recombinant host cell comprising a recombinant PCSK9 gene according to the present invention or a vector as defined above. Suitable host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include  E.coli, Kluyveromyces  or  Saccharomyces  yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.). More particularly, the invention contemplates liver and small intestine and cells thereof or derived thereof.  
         [0056]    The present invention also relates to a method for producing a recombinant host cell expressing a NARC-1 polypeptide comprising an alteration according to the present invention, said method comprising (i) introducing in vitro or ex vivo into a competent host cell a recombinant nucleic acid or a vector as described above, (ii) culturing in vitro or ex vivo the recombinant host cells obtained and (iii), optionally, selecting the cells which express and/or secrete said NARC-1 polypeptide.  
         [0057]    Such recombinant host cells can be used for the production of NARC-1 polypeptides according to the present invention, as well as for screening of active molecules, as described below. Such cells may also be used as a model system to study hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders. These cells can be maintained in suitable culture media, such as DMEM, RPMI, HAM, etc., in any appropriate culture device (plate, flask, dish, tube, pouch, etc.).  
         [0058]    Diagnosis  
         [0059]    The invention now provides diagnosis methods based on a monitoring of alteration at the PCSK9 gene locus in a subject. Within the context of the present invention, the term “diagnosis” includes the detection, monitoring, dosing, comparison, etc., at various stages, including early, pre-symptomatic stages, and late stages, in adults, children and pre-birth. Diagnosis typically includes the prognosis, the assessment of a predisposition or risk of development, the characterization of a subject to define most appropriate treatment (pharmaco-genetics), etc.  
         [0060]    A particular object of this invention resides in a method of detecting the presence of or predisposition to hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders in a subject, the method comprising (i) providing a sample from the subject and (ii) detecting the presence of an alteration in the PCSK9 gene locus in said sample, the presence of said alteration is indicative of the presence of or predisposition to hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders. Preferably, said alteration is a nucleotide substitution. More preferably, the invention concerns a method of detecting the presence of or predisposition to ADH.  
         [0061]    A particular object of this invention resides in a method of detecting the presence of or predisposition to hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders in a subject, the method comprising (i) providing a sample from the subject and (ii) detecting the presence of an alteration in the NARC-1 MRNA in said sample, the presence of said alteration is indicative of the presence of or predisposition to hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders. Preferably, said alteration is a nucleotide substitution. More preferably, the invention concerns a method of detecting the presence of or predisposition to ADH.  
         [0062]    An additional particular object of this invention resides in a method of detecting the presence of or predisposition to hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders in a subject, the method comprising (i) providing a sample from the subject and (ii) detecting the presence of an alteration in the NARC-1 polypeptide in said sample, the presence of said alteration is indicative of the presence of or predisposition to hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders. Preferably, said alteration is an amino acid substitution. More preferably, the invention concerns a method of detecting the presence of or predisposition to ADH.  
         [0063]    An other particular object of this invention resides in a method of assessing the response of a subject to a treatment of hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders, the method comprising (i) providing a sample from the subject and (ii) detecting the presence of an alteration in the PCSK9 gene locus, in the NARC-1 MRNA or in the NARC-1 polypeptide in said sample, the presence of said alteration is indicative of a particular response to said treatment. Preferably, said alteration is a nucleotide or amino acid substitution. More preferably, the invention concerns a method of assessing the response of a subject to a treatment of ADH.  
         [0064]    A further object of the present invention resides in a method of detecting the presence of or predisposition to hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders in a subject, the method comprising (i) providing a sample from the subject and (ii) detecting the presence of an alteration in the PCSK9 gene, the LDL receptor gene and/or the apolipoprotein B gene in said sample, the presence of said alteration is indicative of the presence of or predisposition to hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders. Similarly, the alteration can also be detected at the protein level. More preferably, the invention concerns a method of detecting the presence of or predisposition to ADH.  
         [0065]    An alteration in the gene may be any form of mutation(s), deletion(s), rearrangement(s) and/or insertions in the coding and/or non-coding region of the locus, alone or in various combination(s). Mutations more specifically include point mutations, as disclosed above. In a preferred embodiment of the present invention, the alteration is a nucleotide or amino acid substitution.  
         [0066]    The detection of the presence of an altered PCSK9 gene or an altered NARC-1 MRNA sequence according to the present invention can be performed by sequencing all or part of the PCSK9 gene, polypeptide or RNA, by selective hybridisation or by selective amplification, for instance.  
         [0067]    A more specific embodiment comprises detecting the presence of a polymorphism as disclosed in Table 2 in the PCSK9 gene sequence or NARC-1 mRNA of a subject.  
         [0068]    More particularly, the alteration of the PCSK9 gene locus is detected through an haplotype segregating with the mutation causing ADH, more preferably the haplotype (polymorphisms B (absence of insertion), H, I, M and U of Table 2).  
         [0069]    Preferably, the alteration detected in the PCSK9 gene locus or NARC-1 mRNA is selected from the group consisting of a substitution of the nucleotide T at position 625 and 890 of SEQ ID No 1 and a combination thereof, more preferably a T→A substitution at position 625 of SEQ ID No 1, a T→C substitution at position 890 of SEQ ID No 1 and a combination thereof.  
         [0070]    Alternatively, the alteration detected in the PCSK9 gene locus or NARC-1 mRNA can also be selected from the group consisting of a substitution of the nucleotide A at position 898 and a substitution of the nucleotide C at position 953 of SEQ ID No 1 and a combination thereof, more preferably a A→T substitution at position 898 of SEQ ID No 1, a C→T substitution at position 953 of SEQ ID No 1 and a combination thereof.  
         [0071]    Preferably, the alteration detected in the NARC-1 protein is selected from the group consisting of a substitution of the residue Serine at position 127 of SEQ ID No 2, a substitution of the residue Phenylalanine at position 216 of SEQ ID No 2 and a combination thereof, more preferably a substitution of the residue Serine at position 127 of SEQ ID No 2 by an Arginine (S127R) or a substitution of the residue Phenylalanine at position 216 of SEQ ID No 2 by a Leucine (F216L) or a combination thereof.  
         [0072]    Alternatively, the alteration detected in the NARC-1 protein can also be selected from the group consisting of a substitution of the residue Arginine at position 218 of SEQ ID No 2, a substitution of the residue Arginine at position 237 of SEQ ID No 2 and a combination thereof, more preferably a substitution of the residue Arginine at position 218 of SEQ ID No 2 by a Serine (R218S) or a substitution of the residue Arginine at position 237 of SEQ ID No 2 by a Tryptophane (R237W) or a combination thereof.  
         [0073]    An object of the present invention resides in a method of detecting the presence of or predisposition to hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders in a subject, the method comprising (i) providing a sample from the subject and (ii) detecting the presence of an altered NARC-1 RNA and/or polypeptide expression, the presence of said altered NARC-1 RNA and/or polypeptide expression is indicative of the presence of or predisposition to hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders. More preferably, the invention concerns a method of detecting the presence of or predisposition to ADH.  
         [0074]    An object of the present invention resides in a method of assessing the response of a subject to a treatment of hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders in a subject, the method comprising (i) providing a sample from the subject and (ii) detecting the presence of an altered NARC-1 RNA and/or polypeptide expression, the presence of said altered NARC-1 RNA and/or polypeptide expression is indicative of a particular response to said treatment. More preferably, the invention concerns a method of detecting the presence of or predisposition to ADH.  
         [0075]    Altered RNA expression includes the presence of an altered RNA sequence, the presence of an altered RNA splicing or processing, the presence of an altered quantity of RNA, etc. These may be detected by various techniques known in the art, including by sequencing all or part of the NARC-1 RNA or by selective hybridisation or selective amplification of all or part of said RNA, for instance.  
         [0076]    Altered NARC-1 polypeptide expression includes the presence of an altered polypeptide sequence, the presence of an altered quantity of NARC-1 polypeptide, the presence of an altered tissue distribution, etc. These may be detected by various techniques known in the art, including by sequencing and/or binding to specific ligands (such as antibodies), for instance.  
         [0077]    A further object of the present invention resides in a method of detecting the presence of or predisposition to hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders in a subject, the method comprising (i) providing a sample from the subject and (ii) detecting the presence of an altered NARC-1 activity, the presence of said altered NARC-1 activity is indicative of the presence of or predisposition to hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders. Preferably, said altered NARC-1 activity is a decreased NARC-1 activity. More preferably, the invention concerns a method of detecting the presence of or predisposition to ADH.  
         [0078]    A further object of the present invention resides in a method of assessing the response of a subject to a treatment of hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders in a subject, the method comprising (i) providing a sample from the subject and (ii) detecting the presence of an altered NARC-1 activity, the presence of said altered NARC-1 activity is indicative of a particular response to said treatment. Preferably, said altered NARC-1 activity is a decreased NARC-1 activity. More preferably, the invention concerns a method of detecting the presence of or predisposition to ADH.  
         [0079]    An object of the present invention resides in a method of genotyping at least one polymorphism of the PCSK9 gene, preferably listed in Table 2, comprising (i) providing a sample from the subject and (ii) determining the identity of the allele of said polymorphism in said sample. Preferably, the identity of the allele is determined by performing a hydridization assay, a sequencing assay, a microsequencing assay, an allele-specific amplification assay.  
         [0080]    The present invention also relates to a method of determining the existence of an association between a polymorphism and a disease or disorder, comprising the steps of: (i) genotyping at least one polymorphism of the PCSK9 gene, preferably one listed in Table 2, in a population having said disease or disorder; (ii) genotyping said polymorphism: in a control population; and, (iii) determining whether a statistically significant association exists between said disease or disorder and said polymorphism.  
         [0081]    As indicated above, various techniques known in the art may be used to detect or quantify altered PCSK9 gene or RNA expression or sequence, including sequencing, hybridisation, amplification and/or binding to specific ligands (such as antibodies). Other suitable methods include allele-specific oligonucleotide (ASO), allele-specific amplification, Southern blot (for DNAs), Northern blot (for RNAs), single-stranded conformation analysis (SSCA), PFGE, fluorescent in situ hybridization (FISH), gel migration, clamped denaturing gel electrophoresis, heteroduplex analysis, RNase protection, chemical mismatch cleavage, ELISA, radio-immunoassays (RIA) and immuno-enzymatic assays (IEMA).  
         [0082]    Some of these approaches (e.g., SSCA and CGGE) are based on a change in electrophoretic mobility of the nucleic acids, as a result of the presence of an altered sequence. According to these techniques, the altered sequence is visualized by a shift in mobility on gels. The fragments may then be sequenced to confirm the alteration. Some others are based on specific hybridization between nucleic acids from the subject and a probe specific for wild-type or altered PCSK9 gene or RNA. The probe may be in suspension or immobilized on a substrate. The probe is typically labelled to facilitate detection of hybrids. By “specific hybridization” is intended a hybridization under stringent conditions.  
         [0083]    Some of these approaches are particularly suited for assessing a polypeptide sequence or expression level, such as Northern blot, ELISA and RIA. These latter require the use of a ligand specific for the polypeptide, more preferably of a specific antibody.  
         [0084]    Sequencing can be carried out using techniques well known in the art, using automatic sequencers. The sequencing may be performed on the complete PCSK9 gene or, more preferably, on specific domains thereof, typically those known or suspected to carry deleterious mutations or other alterations.  
         [0085]    Amplification may be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). These techniques can be performed using commercially available reagents and protocols. Preferred techniques use allele-specific PCR or PCR-SSCP. Amplification usually requires the use of specific nucleic acid primers, to initiate the reaction.  
         [0086]    In this regard, a particular object of this invention resides in a nucleic acid primer useful for amplifying sequences from the PCSK9 gene or locus. Such primers are preferably complementary to, and hybridize specifically under stringent conditions to nucleic acid sequences in the PCSK9 gene locus. Particular primers are able to specifically hybridise under stringent conditions with a portion of the PCSK9 gene locus that flank a target region of said locus, said region comprising an alteration according to the present invention, more particularly a substitution of the nucleotide T at position 625 and /or 890 of SEQ ID No 1 or a polymorphism listed in Table 2, preferably said target region being altered in certain subjects having ADH.  
         [0087]    A aspect of this invention includes a pair of nucleic acid primers, wherein said pair comprises a sense and a reverse primers, and wherein said sense and a reverse primers specifically amplify a PCSK9 gene or RNA or a target region thereof, said region comprising an alteration according to the present invention, more particularly a substitution of the nucleotide T at position 625 and /or 890 of SEQ ID No 1 or a polymorphism listed in Table 2, preferably said target region being altered in certain subjects having hypercholesterolemia, more particularly ADH and/or lipid and lipoprotein metabolism disoders.  
         [0088]    In a more specific embodiment, the invention relates to a nucleic acid primer, wherein said primer is complementary to and hybridizes specifically under stringent conditions to a portion of a PCSK9 coding sequence (e.g., gene or RNA), wherein said portion comprising an alteration according to the present invention, more particularly a substitution of the nucleotide T at position 625 and /or 890 of SEQ ID No 1 or a polymorphism listed in Table 2. Preferably, said alteration is present in certain subjects having hypercholesterolemia, more particularly ADH and/or lipid and lipoprotein metabolism disoders. In this regard, particular primers of this invention are specific for altered sequences in a PCSK9 gene or RNA. By using such primers, the detection of an amplification product indicates the presence of an alteration in the PCSK9 gene locus. In contrast, the absence of amplification product indicates that the specific alteration is not present in the sample. More preferably, said primers comprises the nucleotide at position 625 and/or 890 of SEQ ID No 1, or the nucleotide at position 5158 and/or 13539 of SEQ ID No 3. Alternatively, said primers comprises one polymorphism listed in Table 2.  
         [0089]    Typical primers of this invention are single-stranded nucleic acid molecules of about 5 to 60 nucleotides in length, more preferably of about 8 to about 25 nucleotides in length. The sequence can be derived directly from the sequence of the PCSK9 gene locus. Perfect complementarity is preferred, to ensure high specificity. However, certain mismatch may be tolerated.  
         [0090]    A particular detection technique involves the use of a nucleic acid probe specific for wild-type or altered PCSK9 gene or RNA, followed by the detection of the presence of a hybrid. The probe may be in suspension or immobilized on a substrate or support (as in nucleic acid array or chips technologies). The probe is typically labelled to facilitate detection of hybrids.  
         [0091]    In this regard, a particular embodiment of this invention comprises contacting the sample from the subject with a nucleic acid probe specific for an altered PCSK9 gene locus, and assessing the formation of an hybrid. In a particular, preferred embodiment, the method comprises contacting simultaneously the sample with a set of probes that are specific, respectively, for wild type PCSK9 gene locus and for various altered forms thereof. In this embodiment, it is possible to detect directly the presence of various forms of alterations in the PCSK9 gene locus in the sample. Also, various samples from various subjects may be treated in parallel.  
         [0092]    A further particular object of this invention resides in a nucleic acid probe specific for a PCSK9 gene or RNA. Within the context of this invention, a probe refers to a polynucleotide sequence which is complementary to and capable of specific hybridisation under stringent conditions with a (target portion of a) PCSK9 gene or RNA, and which is suitable for detecting polynucleotide polymorphisms, preferably the polymorphism associated with PCSK9 alleles which predispose to or are associated with ADH. Probes are preferably perfectly complementary to the PCSK9 gene, RNA, or target portion thereof. Probes typically comprise single-stranded nucleic acids of between 8 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. It should be understood that longer probes may be used as well. A preferred probe of this invention is a single stranded nucleic acid molecule of between 8 to 500 nucleotides in length, which can specifically hybridise under stringent conditions to a region of a PCSK9 gene or RNA that carries an alteration.  
         [0093]    A specific embodiment of this invention is a nucleic acid probe specific for an altered (e.g., a mutated) PCSK9 gene or RNA, i.e., a nucleic acid probe that specifically hybridises under stringent conditions to said altered PCSK9 gene or RNA and essentially does not hybridise under stringent conditions to a PCSK9 gene or RNA lacking said alteration. Specificity indicates that hybridisation to the target sequence generates a specific signal which can be distinguished from the signal generated through non-specific hybridisation. Perfectly complementary sequences are preferred to design probes according to this invention. It should be understood, however, that certain mismatch may be tolerated, as long as the specific signal may be distinguished from non-specific hybridisation.  
         [0094]    Particular examples of such probes are nucleic acid sequences complementary to a target portion of the PCSK9 gene or RNA carrying the nucleotide at position 625 and/or 890 of SEQ ID No 1, the nucleotide at position 5158 and/or 13539 of SEQ ID No 3, a polymorphism listed in Table 2, or a mutation disclosed in Table 4.  
         [0095]    The sequence of the probes can be derived from the sequences of the PCSK9 gene and RNA as provided in the present application. Nucleotide substitutions may be performed, as well as chemical modifications of the probe. Such chemical modifications may be accomplished to increase the stability of hybrids (e.g., intercalating groups) or to label the probe. Typical examples of labels include, without limitation, radioactivity, fluorescence, luminescence, enzymatic labelling, etc.  
         [0096]    As indicated above, alteration in the PCSK9 gene locus may also be detected by screening for alteration(s) in NARC-1 polypeptide sequence or expression levels. In this regard, a specific embodiment of this invention comprises contacting the sample with a ligand specific for an altered NARC-1 polypeptide and determining the formation of a complex.  
         [0097]    Different types of ligands may be used, such as specific antibodies. In a specific embodiment, the sample is contacted with an antibody specific for an altered NARC-1 polypeptide and the formation of an immune complex is determined. Various methods for detecting an immune complex can be used, such as ELISA, radio-immunoassays (RIA) and immuno-enzymatic assays (IEMA).  
         [0098]    In a specific embodiment, the method comprises contacting a sample from the subject with (a support coated with) an antibody specific for an altered form of a NARC-1 polypeptide, and determining the presence of an immune complex. In a particular embodiment, the sample may be contacted simultaneously, or in parallel, or sequentially, with various (supports coated with) antibodies specific for different forms of a NARC-1 polypeptide, such as a wild-type and various altered forms thereof.  
         [0099]    Particular examples of such specific ligands are antibodies specific for altered NARC-1 polypeptide sequence resulting from any mutation in position 127 and/or 216, more particularly a substitution of the residue Serine at position 127 by an Arginine (S 127R) or a substitution of the residue Phenylalanine at position 216 by a Leucine (F216L) or any combination of those mutations.  
         [0100]    The invention also relates to a diagnostic kit comprising products and reagents for detecting in a sample from a subject the presence of an alteration in the PCSK9 gene or in the NARC-1 protein, in the NARC-1 RNA or polypeptide expression, and/or in NARC-1 activity. Optionally, said diagnostic kit further comprises reagents for detecting in a sample from a subject the presence of an alteration in the LDL receptor and/or the apolipoprotein B. Said diagnostic kit according to the present invention comprises any primer, any pair of primers, any nucleic acid probe and/or any antibody described in the present invention. Said diagnostic kit according to the present invention can further comprise reagents and/or protocols for performing a hybridization, amplification or antigen-antibody immune reaction.  
         [0101]    SCREENING  
         [0102]    The present invention also provides novel targets and methods for the screening of drug candidates or leads. Such drug candidates or leads are useful for developping a treatment against hypercholesterolemia, more particularly ADH, lipid and lipoprotein metabolism disorders, atherosclerosis, and/or CVD. Preferably, such drug candidates or leads are useful for developping a treatment against ADH. The methods include binding assays and/or functional assays, and may be performed in vitro, in cell systems, in animals, etc. Functional assays comprise, but are not limited to, the cleavage of a substrate. The in vitro assays, cell-based assays and animal-based assays involve a NARC-1 protein, preferably a NARC-1 protein comprising an alteration according to the present invention. Optionally, said assays comprise a control with a natural NARC-1 protein.  
         [0103]    For cell systems, cells can be native, i.e., cells that normally express the NARC-1 polypeptide, as a biopsy or expanded in cell culture. Preferably, these native cells are derived from liver or small intestine. Alternatively, cells are recombinant host cells expressing NARC-1, more particularly a NARC-1 protein comprising an alteration according to the present invention.  
         [0104]    The invention relates to methods for identifying of the target proteins of the NARC-1 protein, preferably a NARC-1 protein comprising an alteration according to the present invention.  
         [0105]    The invention relates to methods for screening of compounds that modulate the NARC-1 activity. Such compounds, for example, can increase or decrease affinity and/or rate of binding of the NARC-1 protein to the substrate, compete with substrate for binding to the NARC-1 protein, or displace substrate bound to the NARC-1 protein. Preferably, the invention concerns methods for screening of compounds that increase or restore the natural NARC-1 activity. By “natural” NARC-1 activity is intended the activity of the wild-type NARC-1 protein. Furthermore, the invention concerns methods for screening of compounds that inhibit the activity of the altered NARC-1 comprising an alteration changing the substrate specificity and, thereby generating new substrates. Said compounds are able to block the activity of the altered NARC-1 for its new substrate.  
         [0106]    Therefore, the present invention concerns a method of selecting biologically active compounds, said method comprising contacting a test compound with an altered PCSK9 gene or an altered NARC-1 protein or fragment thereof of at least 15 consecutive residues comprising an alteration, wherein the alteration reduces, modifies, or abolishes the activity of NARC-1, and determining the ability of said test compound to modulate the expression and/or activity of said gene or protein or fragment.  
         [0107]    A particular object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a PCSK9 gene or NARC-1 polypeptide, preferably a PCSK9 gene or a NARC-1 polypeptide, or a fragment thereof of at least 15 consecutive residues, comprising an alteration according to the present invention, and determining the ability of said test compound to bind said PCSK9 gene or NARC-1 polypeptide. Binding to said gene or polypeptide provides an indication as to the ability of the compound to modulate the activity of said target, and thus to affect a pathway leading to hypercholeterolemia, more particularly ADH, and lipid and/or lipoprotein metabolism disorders in a subject. In a preferred embodiment, the method comprises contacting in vitro a test compound with a NARC-1 polypeptide or a fragment thereof, preferably a NARC-1 polypeptide or a fragment thereof comprising an alteration according to the present invention, and determining the ability of said test compound to bind said NARC-1 polypeptide or fragment. The fragment preferably comprises a substrate-binding site of the NARC-1 polypeptide.  
         [0108]    A particular object of this invention resides in a method of selecting compounds active against hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders, said method comprising contacting in vitro a test compound with a NARC-1 polypeptide or a fragment thereof of at least 15 consecutive residues, preferably a NARC-1 polypeptide or a fragment thereof comprising an alteration according to the present invention, and determining the ability of said test compound to bind said NARC-1 polypeptide or fragment thereof. The NARC-1 polypeptide or fragment thereof may be used in essentially pure form, in suspension, or immobilized on a support.  
         [0109]    In a futher particular embodiment, the method comprises contacting a recombinant host cell expressing NARC-1 polypeptide, preferably a NARC-1 polypeptide comprising an alteration according to the present invention, with a test compound, and determining the ability of said test compound to bind said NARC-1 polypeptide and/or to modulate the activity of NARC-1 polypeptide.  
         [0110]    The determination of binding may be performed by various techniques, such as by labelling of the test compound, by competition with a labelled reference ligand, two-hybrid Screening Assay, etc. Modulation of activity includes, without limitation, the inhibition or activation of the autocatalytic processing of pro-NARC-1, and/or the inhibition or activation of the substrate cleavage, more particularly a synthetic substrate comprising a zymogenic processing site.  
         [0111]    A further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a NARC-1 polypeptide, preferably a NARC-1 polypeptide comprising an alteration according to the present invention, and determining the ability of said test compound to modulate the activity of said NARC-1 polypeptide.  
         [0112]    A further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a PCSK9 gene, preferably a PCSK9 gene comprising an alteration according to the present invention, and determining the ability of said test compound to modulate the expression of said PCSK9 gene.  
         [0113]    The invention also concerns methods of selecting biologically active compounds using a non-human transgenic animals expressing a NARC-1 protein, preferably a NARC-1 protein comprising an alteration according to the present invention. Optionally, said non-human transgenic animals can be homozygote or heterozygote for the altered PCSK9 gene. Said methods comprise (i) administrating a test compound to said non-human transgenic animal, and (ii) determining the ability of said test compound to modulate the NARC-1 activity. Said NARC-1 activity can be assessed by determining the plasmatic concentration of cholesterol and/or lipoparticules (VLDL, IDL, LDL), by determining the plasmatic enzymatic activity of NARC-1, by analyzing some tissues (liver, small intestine), by determining the lipoprotein kinetics. The enzymatic activity of NARC-1 can be determined with synthetic substrate, such as described in Seidah et al (2003).  
         [0114]    The above screening assays may be performed in any suitable device, such as plates, tubes, dishes, flasks, etc. Typically, the assay is performed in multi-wells plates. Several test compounds can be assayed in parallel.  
         [0115]    Furthermore, the test compound may be of various origin, nature and composition. It may be any organic or inorganic substance, such as a lipid, peptide, polypeptide, nucleic acid, small molecule, etc., in isolated or in mixture with other substances. The compounds may be all or part of a combinatorial library of products, for instance. The test compounds can be an antisense or an RNAi. The test compounds can be competitive or suicide substrates. By “suicide substate” is intended a compounds that, after binding NARC-1 protein, the reactive group forms an irreversible bond with NARC-1 rendering it inactive.  
         [0116]    Therapy  
         [0117]    The invention contemplates methods of treatment of hypercholesterolemia, more particularly ADH, lipid and lipoprotein metabolism disorders, atherosclerosis and/or CVD. Preferably, the invention relates to methods of treatment of hypercholesterolemia, more particularly ADH, and/or lipid and lipoprotein metabolism disorders due to an alteration of NARC-1 protein.  
         [0118]    The invention also relates to a method of treating or preventing hypercholesterolemia, more particularly ADH, lipid and lipoprotein metabolism disorders, atherosclerosis and/or CVD in a subject, the method comprising administering to said subject a functional (e.g., wild-type) NARC-1 polypeptide or a nucleic acid encoding the same. More preferably, the invention concerns a method of treating or preventing ADH.  
         [0119]    The invention concerns the use of a functional NARC-1 polypeptide or a nucleic acid encoding the same, in the manufacture of a pharmaceutical composition for treating or preventing hypercholesterolemia, more particularly ADH, lipid and lipoprotein metabolism disorders, atherosclerosis and/or CVD in a subject. More preferably, the invention concerns a pharmaceutical composition for treating or preventing ADH.  
         [0120]    The invention also relates to a method of treating or preventing hypercholesterolemia, more particularly ADH, lipid and lipoprotein metabolism disorders, atherosclerosis and/or CVD in a subject, the method comprising administering to said subject a compound that modulates NARC-1 expression and/or activity. More preferably, the invention concerns a method of treating or preventing ADH. The invention further relates to a pharmaceutical composition comprising a compound that modulates NARC-1 expression and/or activity.  
         [0121]    The invention relates, generally, to the use of a compound that modulates NARC-1 expression and/or activity in the manufacture of a pharmaceutical composition for treating or preventing hypercholesterolemia, more particularly ADH, lipid and lipoprotein metabolism disorders, atherosclerosis and/or CVD in a subject. More preferably, the invention concerns a pharmaceutical composition for treating or preventing ADH.  
         [0122]    The present invention demonstrates the causal link between hypercholesterolemia, more particularly ADH, and an alteration of the PCSK9 gene locus. The invention thus provides a novel target of therapeutic intervention. Various approaches can be contemplated to restore or modulate the NARC-1 activity or function, more particularly normal NARC-1 activity or function, in a subject, particularly those carrying an altered PCSK9 gene locus. Supplying wild-type function to such subject is expected to suppress phenotypic expression of hypercholesterolemia, more particularly ADH, in a pathological cell or organism. The supply of such function can be accomplished through gene or protein therapy, or by administering compounds that modulate NARC-1 activity.  
         [0123]    If the alteration of NARC-1 protein leads to a decrease or loss of NARC-1 activity, the treatment consists in administering a biologically active compound which increases or restores the NARC-1 activity. Said biologically active compound can be a natural NARC-1 protein. Alternatively, said compound can be an activator of the NARC-1 protein. Said compound can also increase the expression of NARC-1 protein.  
         [0124]    If the alteration of NARC-1 protein leads to a new specificity for a substrate, the treatment consists in administrating a biologically active compound which inhibits the activity of the altered NARC-1 protein. Said compound can decrease the expression of NARC-1 protein. For example, such compounds can be an antisens or an RNAi of PCSK9 gene comprising the alteration causing ADH. Alternatively, said compound can be an inhibitor of the altered NARC-1 protein. Said compound can compete with the substrate or can be a suicide substrate.  
         [0125]    Further aspects and advantages of the present invention will be disclosed in the following experimental section, which should be regarded as illustrative and not limiting the scope of the present invention. The references cited in the present application are all incoporated herein by reference.  
       EXAMPLE  
       [0126]    The inventors mapped a third locus HCHOLA3 at 1p32 and now report two mutations in the PCSK9 gene causing ADH. PCSK9 encodes NARC-1 (neural apoptosis regulated convertase). Its mutations lead to reduced activation of the enzyme. Lipoprotein kinetics in probands revealed an overproduction of apoB100-rich particles showing that the pathogenic origin of the disease is hepatic. In conclusion, NARC-1 is a newly identified human subtilase that contributes to cholesterol homeostasis and is the first example of a dominant disease associated with a defect in a member of the large subtilase family.  
         [0127]    To identify the HCHOLA3 locus (formerly FH3), that the inventors mapped (Varret et al, 1999) to 1p34.1-p32 (OMIM603776) and was confirmed by Hunt et al. in a large Utah kindred (Hunt et al, 2000), the inventors performed positional cloning using the originally linked family and 23 French families in which the implication of the LDLR and APOB genes had been excluded.  
         [0128]    Family HC92 was identified through the proband (HC92-II-7) who belongs to a multiplex ADH pedigree from which twenty-nine family members were sampled and tested in parametric linkage analyses. In the reduced pedigree studied in the linkage analysis, 12 subjects presented with total cholesterol levels above the 97.5 th percentile when compared with other French individuals matched by age and sex (Steinmetz, 1990) (mean total cholesterol: 3.63 g/L ±0.68, mean LDL-cholesterol: 2.87±0.72 g/L). The inventors excluded linkage to the LDLR and APOB genes [lod scores at −14.05 and −10.01 (θ=0.0), respectively]. The family was genotyped for 8 Genethon markers in the 1p34-p32 region (FIG. 1). The inventors obtained highly significant lod scores with a maximum of 4.26 (θ=0.0) at D1S2742 that reached 4.80 in the multipoint analyses (Table 1, FIG. 3 a ). Haplotype analysis identified a 5.9 Mb critical interval between D1S231 and D1S2890. The critical interval that our team had previously reported in the HC2 family (Varret et al, 1999) was between markers D1S472 and D1S211, thus more distal. Reexamination of haplotype data (FIG. 2) showed that all affected subjects of the HC2 family also shared the same haplotype between markers D1S2722 and D1S2890 except HC2-II-5. This “affected” subject presented a recombinational event at D1S211 thus providing the centromeric boundary of the region described in 1999. Therefore all family members were reinvestigated. HC2-II-5 (who refuses treatment) was the only subject who showed a significant variation (a marked elevation of triglycerides) and thus no longer conforms with the inclusion criteria.  
         [0129]    The inventors established the physical map of the candidate region between D1S197 and D1S2890 covered by 82 overlapping BAC sequences released from the Human Genome Project. The region between D1S197 and D1S2890 contains 41 genes among which 8 encode interesting functional candidates with respect to lipid metabolism: EPS 15 (Epidermal growth factor receptor pathway substrate-15), OSBPL9 (Oxysterol binding protein-like 9), SCP2 (Sterol carrier protein 2), LRP8 (Low density lipoprotein receptor-related protein 8), DHCR24 (24-dehydrocholesterol reductase), PRKAA2 (Protein kinase, AMP-activated, alpha 2 catalytic subunit), DAB1 (Disabled homolog 1) and PCSK9 (encoding NARC-1). This Neural Apoptosis Regulated Convertase 1 is a novel putative proprotein convertase (PC) belonging to the subtilase subfamily (Seidah et al, 2003). A related protein is the subtilisin kexin isoenzyme-1 (SKI-1)/site-1-protease (SIP) known to play a key role in cholesterol homeostasis through the processing of the sterol regulatory element-binding proteins (SREBPs) (Brown &amp; Goldstein, 1999; Elagoz et al, 2002). The cDNA spans 3617 bp encoding a 692 amino acid protein. NARC-1 was mapped to 1p33-p34.3. The inventors precisely localized its cDNA using the Blast program (http://www.ncbi.nlm.nih.gov/BLAST/) in the HCHOLA3 interval as follows: tel-D1S231-D1S2661-D1S417-D1S2652-PCSK9-D1S475-D1S200-D1S2742-cen.  
         [0130]    Systematic bidirectional sequencing of the 125 exons of the first seven candidates revealed no mutation in probands. By sequencing the 12 exons of PCSK9 the inventors identified in family HC92 a T→A substitution in exon 2 at nucleotide 625 predicting a substitution at codon 127 of Arginine for the conserved Serine (S127R), thereby creating a MnlI cleavage site (FIG. 3 b , FIG. 4). HC92 family members and 100 controls were tested for the substitution. It was absent in the 200 control chromosomes indicating that it is not a polymorphism. It was found in the 12 affected family members and in subject HC92-IV-3 who has a total cholesterol level in the  90 th percentile when compared to other French individuals matched by age and sex. Thus, the penetrance in the family is estimated at 0.94. Interestingly, the S127R mutation was also found in the proband of HC2 and cosegregated with the disease in the family except in subject HC2-II-5, confirming that he had been misclassified in the linkage analyses previously reported (Varret et al, 1999). To assess the possible recurrence of this mutation, the inventors tested 5 intragenic polymorphic markers that the inventors had identified in PCSK9 (4 SNPs and a GCT repeat) in both families. The same haplotype segregated with the S127 R mutation in both the HC2 and the HC92 family: (Polymorphisms B (absence of insertion), H, I, M, and U) (Tables 2 and 3). Furthermore, a unique haplotype was also obtained for the extragenic markers surrounding PCSK9 (D1S2661, D1S417, D1S475, D1S200 and D1S2742) in both families. These results show that despite the absence of records and different geographical origins, the families share a common ancestor. The possibility of a French founder effect was ruled out since the mutation was not found in 22 other French ADH probands.  
         [0131]    Through systematic bidirectional sequencing of the 12 exons of the PCSK9 gene in 22 ADH probands, a second mutation (F216L) was identified in the proband of the HC60 family (FIG. 2 c ) who died from myocardial infarction at 49 y.o (FIG. 3 d , FIG. 4). This mutation segregated with the ADH phenotype in the family and was not found in 200 control chromosomes. No major rearrangement was found in any of the probands by Southern blot (data not shown). Thus, mutations in PCSK9 have been found in 12.5% of the ADH families tested.  
         [0132]    The inventors also identified 25 polymorphisms present in different probands and on control chromosomes from subjects with normal cholesterol levels (Table 2). These variations and their respective frequencies in the French population are listed in Table 2. It should be noted that none of these polymorphisms give rise to new donor or acceptor splice sites (score calculated according to Senapathy et al.) (Senapathy et al, 1990; Shapiro &amp; Senapathy, 1987).  
         [0133]    In order to unravel at the molecular level the consequences of the S127R and F216L mutations, the inventors introduced them in the human PCSK9 cDNA (Seidah et al., 2003). The inventors also obtained four other mutants, namely S127A, S127P, 15 — 16insL (polymorphic variant where an extra leucine is added in the signal peptide hydrophobic stretch) and the active site mutant H226A (Seidah et al., 2003). The cDNAs encoding wild type (WT) NARC-1 and its mutants containing a C-terminal V5 epitope, were transiently transfected in HEK293 cells. A 4h pulse with  35 S-labelled Met and Cys was followed by immunoprecipitation of the cell lysates and the media with a V5 mAb (Seidah et al., 2003). The inventors have previously shown that proNARC-1 is synthesized as a 72 kDa precursor that undergoes two zymogen cleavage events. The first one is rapid and occurs in the endoplasmic reticulum (ER) at the YVVVL 82 ⇓ site, giving rise to the 63-65 kDa N1 product and the 14 kDa prosegment (pro). The second one occurs with much lower efficacy at the putative PHVDY 142 ⇓ site and gives rise to the presumably active 58 kDa N2 enzyme (Brown &amp; Goldstein, 1999). By STORM quantitation the inventors estimate that both S127R and F216L mutations lead to ˜3-fold lower levels of N2. In addition, the secreted level of N1 was about 2-fold lower for the S127R mutant. Interestingly, while the S127A mutant shows a similar behavior, the S127P resembles WT. Finally, the 15 — 16insL allelic variant seems to give rise to a ˜2-fold higher percentage of NI and N2 products, suggesting that more active NARC-1 is produced.  
         [0134]    The inventors have identified a new gene implicated in ADH by positional cloning. Linkage analyses were performed on two large French pedigrees: HC92 and HC2 in which the implication of the LDLR and APOB genes had been excluded. A maximum lod score of 4.26 was obtained for D1S2742 in family HC92. Haplotype analysis restricted the region of linkage to a 5.9 Mb interval between markers D1S231 and D1S2890 at 1p32. Our team had previously reported the localization of HCHOLA3 at 1p32-p34.1 by. linkage analysis performed on the HC2 family (Varret et al, 1999). In this family, the critical interval was flanked by markers D1S472 and D1S211 and was thus more distal as compared to the one identified with the HC92 family. Reexamination of haplotype data showed that all affected subjects of the HC2 family also shared the same haplotype between markers D1S2722 and D1S2890 except (HC2-II-5). This “affected” subject presented a recombinational event at D1S211 thus providing the centromeric boundary of the region described in 1999. Therefore (HC2-II-5) was reinvestigated. The new lipid measurements showed the same elevated cholesterol but also marked elevation of triglycerides. This alteration can be explained by recent knowledge of a notable alcohol intake that presently prohibits proper assessment of the subject&#39;s status with respect to the family trait. Identification of the S127R PCSK9 gene mutation in all other affected members of the HC2 family and its absence in (HC2-II-5) confirmed that he had been misclassified for the genetic analyses. Identification of the S127R PCSK9 gene mutation in the HC92 family also helped to clarify the genetic status of the 8 children that had been sampled but not included in the linkage analyses. These results comforted the conservative approach that the inventors had chosen (total cholesterol above the  97 . 5 th percentile when compared with sex- and age-matched French population) and that also allowed for reduced penetrance. This last parameter was confirmed since the S127R PCSK9 gene mutation was identified in (HC92-IV-3) who has a total cholesterol level in the 90 th  percentile (when compared to other French individuals matched by age and sex), and had higher cholesterol levels when compared to the levels of his non-affected sisters (2.5 th  percentile for HC92-IV-1 and 30 th  percentile for HC92-IV-2). Thus, the penetrance can now be estimated at 0.94 in the family when considering the inclusion criteria that were applied. This characteristic of a PCSK9 gene mutation is also found with LDLR gene mutations (Hobbs et al, 1989; Sass et al, 1995) and more generally accounts for the variability of the hypercholesterolemic phenotype (evaluated by common clinical and biological criteria) that can be due to the effect of environmental factors or of modifier genes.  
         [0135]    Haplotype analysis showed that a unique haplotype segregated with the S127R mutation in both the HC92 and HC2 families. Therefore, it can be assumed that despite the absence of records and different geographical origins, the families share a common ancestor. The possibility of a French founder effect can be ruled out since the mutation was not found in a total of 22 other French probands (data not shown).  
         [0136]    NARC-1 is a novel convertase recently cloned by two pharmaceuticals companies (NARC-1, Millenium Pharmaceuticals and LP251, Eli Lilly). It was first identified via the cloning of cDNAs upregulated following apoptosis induced by serum deprivation in primary cerebellar neurons. NARC-1 was more precisely characterized recently by Seidah et al. who used short conserved segments of the SKI-1 catalytic subunit as baits and the Protein Blast program to identify this convertase in a patented database (Seidah et al, 2003). It is synthesized as a soluble zymogen that undergoes autocatalytic intramolecular processing in the endoplasmic reticulum (ER) at the primary cleavage site YVVVL⇓KEE 85  indicative of the enzymatic specificity (Seidah et al, 2003) of NARC-1. Prosegment cleavage is necessary for NARC-1 exit from the ER. The S127R mutation resides between the primary and putative secondary zymogen processing sites of proNARC-1, while F216L is located close to the active site (H226). Notably, the S127R mutation creates an RGD site that may be involved in integrin binding (Ruoslahti, 1996).  
         [0137]    While only the S127R mutant causes reduction in the secreted level of N1, both the S127R and the F216L mutations result in reduced production of the enzymatically active N2. Furthermore, the kinetics of VLDL, IDL and LDL apo B 100 performed in ADH subjects carrying the S127R mutation showed an overproduction from the liver of apo B100-rich lipoproteins. Thus, the dominance of the disease shows that NARC-1 is a rate-limiting enzyme involved in cholesterol homeostasis in the liver. Although most enzymopathies are recessively inherited, dominance is reported in some highly regulated or tissue specific enzymes. This is observed in two types of porphyria: AIP (acute intermittent porphyria) (Desnick et al, 1985) and PCT (porphyria cutanea tarda) (Felsher et al, 1982), that are caused by a porphobilinogen deaminase and uroporphyrinogen decarboxylase deficiency, respectively. However, contrary to porphyria, PCSK9 gene defects seem highly penetrant. NARC-1 belongs to the 9-membered mammalian subtilase family in which only one other member was known to carry a disease-causing mutation: a compound heterozygosity in the PC1 gene results in obesity and endocrinopathy due to impaired prohormone processing (MIM 162150). However, heterozygosity for one of the mutations is silent thus suggesting a recessive transmission (Jackson et al, 1997). Although PCs activate a wide variety of proteins, it is notable that none of them was linked so far to a dominant human disease. NARC-1 is thus unique in this respect and may lead to the discovery of others. While the related convertase SKI-1/S1P plays a key role in regulating cholesterol and fatty acid homeostasis through the processing of SREBP1 and SREBP2, the precise implication of NARC-1 in cholesterol homeostasis is still under investigation. Interestingly, NARC-1 is mainly expressed in the liver and small intestine both of which play key roles in cholesterol synthesis and regulation (Seidah et al, 2003). Since apo B 100 levels are regulated post-translationally (Bostrom et al, 1988), it is possible that NARC-1 could inactivate apo B100 and hence decrease the level of LDL. Indeed, a putative site LIEIGL⇓EGK 668  of apo B100 is proposed which would respect the primary and secondary structure requirements of NARC-1 processing selectivity (Seidah et al, 2003). This may thus explain the reported 70 kDa form of apo B100 that is observed to occur under stressful cellular conditions (Cavallo et al, 1999).  
         [0138]    The crucial role of NARC-1 is revealed by the hypercholesterolemia that occurs when the gene is mutated resulting in a decreased NARC-1 activation. The identification of NARC-1 substrate(s) will help to elucidate novel disease mechanisms and constitute a target(s) for new intervention strategies to limit elevation of LDL particles and prevent morbidity and mortality from premature atherosclerosis.  
         [0139]    Methods  
         [0140]    Family Recruitement  
         [0141]    The French hypercholesterolemic families were recruited through the 8 lipid clinics of the National Network for ADH (“Réseau National de Recherche sur les Hypercholestérolémies Familiales”). Probands were ascertained among consecutive patients of the clinics. Inclusion criteria for probands were: total cholesterol above the 97.5 th  percentile when compared with sex- and age-matched French population (Steinmetz, 1990), LDL cholesterol above 1.9 g/L or 1.6 g/L for children, triglycerides below 1.5 g/L, personal or documented familial xanthomas, and/or arcus corneae, and early CVD. Lipid measurements were repeated to ascertain the existence of primary isolated hypercholesterolemia due to elevated LDL. Family history and pedigrees were investigated. Informed consent was obtained for all subjects included in this study. Family HC2 has been previously reported and described at length (Varret et al, 1999). Functional tests showed normal binding, internalization and degradation of LDL particles in fibroblasts from the probands (HC2-II-9) (Hobbs et al, 1989). Five other families (HC35, HC60, HC92, HC122, HC243) were studied representing 26 affected and 26 unaffected subjects. For affected subjects, mean total and LDL cholesterol were 3.27 g/L ±0.77 and 2.47±0.76 g/L, respectively.  
         [0142]    DNA Analysis and Genotyping  
         [0143]    DNA was isolated from whole blood samples as previously described (Collod et al, 1994). All families were tested with polymorphic markers of the LDLR and APOB genes. For the LDLR, two intragenic markers (D19S584 in intron 1 and the (TA)n in exon 18) and two flanking markers (D19S394 and D19S221) were studied. The 5′HVR (TG repeat) and 3′HVR (VNTR) were studied for the APOB gene and screening for the R3500Q mutation as reported (Rabes et al, 1997). Genotyping at 1p34-p32 was performed using 11 microsatellites from the Genethon map (D1S472, D1S2722, D1S211, D1S197, D1S231, D1S2661; D1S417, D1S475, D1S200, D1S2742, D1S2890) as reported (Collod et al, 1994).  
         [0144]    Linkage Analysis  
         [0145]    Parametric linkage analyses were performed with accepted parameters of ADH: dominant transmission of the trait, penetrance of 0.9 for heterozygotes, and a frequency of the disease allele of 1/500. The MLINK and LINKMAP programs (Ott, 1991), and the VITESSE program (O&#39;Connell &amp; Weeks, 1995) were used to perform the two-point and multipoint LOD score analyses. Microsatellite allele frequencies were calculated among the unrelated family members. Linkage was investigated with the assumption of equal female-to-male recombination rates.  
         [0146]    Candidate Gene Identification and Analysis  
         [0147]    Microsatellites of the 1p34-p32 region were localized on sequences of the Human Genome Project, a physical map of the region was in agreement with the one published by UCSC: http://www.genome.ucsc.edu. Repeat Masker and Genscan programs allowed the prediction and the identification in the Genbank database of positional candidate genes. The Blast program (http://www.ncbi.nlm.nih.gov/BLAST/) was used to localize precisely the candidate genes. The intron/exon structure of the 8 finctional candidates was determined and primers designed with the Mac Vector® software. 137 primer pairs were chosen at approximatively 100 bp surrounding each exon boundary. PCRs were performed with thermostable DNA polymerase from LAROVA Biochemic GmbH (Germany) on GeneAmp® PCR system 9600 (Perkin Elmer). Fluorescent sequencing was carried-out with Big Dye Terminator version 1.0 on GeneAmp® PCR system 9700 (Perkin Elmer) apparatus, under conditions supplied by the manufacturer. Electrophoregrams were analyzed using Sequencing Analysis®3.4 and SeqED®.  
         [0148]    PCSK9 Analysis  
         [0149]    Primers designed to study the 12 exons of NARC-1, and their conditions of amplification are available on request. Major rearrangements for NARC-1 were investigated by Southern blot as reported (Collot et al, 1994). A rapid detection method of the S127R mutation using PCR amplification followed by digestion by Mnll was developped. After amplification of exon 2, the 543 bp PCR product was digested by 5U MnlI enzyme. After electrophoresis on a 2% agarose gel, fragments of 208, 203 and 60 bp were distinguished in the normal allele, while fragments of 208, 143 and 60 bp appeared in the mutated alleles (the 203 bp normal fragment was divided in fragments of 143 and 60 bp and the two 60 bp fragments generated comigrated). Segregation analysis of this mutation in families HC2 and HC92 and analysis of 200 chromosomes from unaffected persons of French descent were tested both by sequencing and by the Mnll -digestion.  
         [0150]    NARC-1 Mutants and Protein Studies  
         [0151]    HEK293 cells were transiently transfected with pIRES2 recombinant vectors (Seidah et al, 2003) expressing wild type hNARC-1-V5 (WT) or its mutants H226A, F216L, S127R, S127A, S127P and 15 — 16insL (+L). 24h later the cells were pulse-labeled with [ 35 S] EasyTag Express mix for 4h. Cell extracts and media were immunoprecipitated with a V5 antibody and the precipitates resolved by SDS-PAGE on an 8% Glycine gels.  
         [0152]    Accession Numbers for the Genes Tested  
         [0153]    EPS15, NM 13  001981; OSBPL9, NM — 024586; SCP2, NM — 002979; LRP8, NM — 004631; DHCR24, NM — 014762; PRKAA2, NM — 006252; DABI, NM — 021080); NARC-1: human AX207686 (gi:15422368); Mus musculus: AX207688; Rattus norvegicus AX207690.  
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         [0171]    Goldstein, J L. &amp; Brown, M S.,  Johns Hopkins Med. J  143, 8-16 (1978).  
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         [0173]    Hunt, S C. et al .  Arterioscler. Thromb. Vasc. Biol.  20, 1089-1093 (2000).  
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         [0175]    Jackson, R. S. &amp; al.  Nature Genet.  16, 303-306 (1997).  
         [0176]    Khachadurian, A K.,  Am. J Med.  37, 402-407, (1964).  
         [0177]    Lusis, A J. Atherosclerosis.  Nature.  407, 233-241 (2000).  
         [0178]    Maugeais, C., Ouguerram, K., Mahot, P., Krempf, M. &amp; Magot, T.,  Diabetes Metab.  22, 57-63 (1996).  
         [0179]    Maugeais, C., Ouguerram, K., Krernpf, M. &amp; Magot, T.  Clin. Chem. Lab. Med.  36, 739-745 (1998).  
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         [0181]    O&#39;Connell, J R. &amp; Weeks D E.  Nature Genet.  11, 402-408 (1995)  
         [0182]    Ott, J. Analysis of human genetic linkage, revised ed. The Johns Hopkins University Press, Baltimore and London (1991)  
         [0183]    Perusse, L.  Arteriosclerosis.  9, 308-318 (1989).  
         [0184]    Pont, F., Duvillard, L., Verges, B. &amp; Gambert, P.  Arterioscier. Thromb. Vasc. Biol.  18, 853-860 (1998).  
         [0185]    Rabes, J P. et al.  Hum. Mutat.  10: 160-163 (1997).  
         [0186]    Rice, T., Vogler, G P., Laskarzewski, P M., Perry, T S. &amp; Rao, D C.  Hum. Biol.  63, 419-439 (1991).  
         [0187]    Ruoslahti, E.  Annu. Rev. Cell. Dev. Biol.  12, 697-715 (1996).  
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         [0194]    Varret, M. et al.  Am. J. Hum. Genet.  64, 1378-1387 (1999). 
         [0195]    [0195]                                                                                                                             TABLE 1                           Regional lod scores obtained in the HC92 family                LOD score at θ            Locus   Distance 1     0.00   0.001   0.01   0.05   0.10   0.20   0.30   0.40   Z max     θ max                      D1S2722   . . .   2.17   2.17   2.13   1.96   1.75   1.31   0.87   0.44   2.17   0.00       D1S211   0.025   −0.77   1.53   2.46   2.86   2.77   2.22   1.48   0.67   2.86   0.05       D1S197   0.063   −1.73   −1.72   −1.62   −1.20   −0.86   −0.48   −0.26   −0.11   −0.11   0.40       D1S231   0.014   1.08   1.08   1.08   1.06   1.00   0.81   0.57   0.30   1.08   0.00       D1S417   0.036   3.12   3.11   3.08   2.89   2.61   1.93   1.16   0.43   3.12   0.00       D1S200   0.007   2.50   2.49   2.45   2.25   1.98   1.44   0.87   0.33   2.50   0.00       D1S2742   0.007   4.26   4.26   4.21   3.97   3.61   2.67   1.79   0.77   4.26   0.00       D1S2890   0.013   0.84   1.29   2.08   2.54   2.52   2.05   1.35   0.58   2.54   0.05                            
         [0196]    [0196]                                                                                   TABLE 2                           Polymorphisms identified in the PCSK9 gene                        Position                           Polymorp.   Nucleotidic   SEQ ID   Amino acid   Nb of indiv.       Exon   Name   Variation   N o 3   variation   tested   Frequency                    1   A   C→T    916   5′UTR   113   0.119               B   Leu stretch   1022-1042   15_16insL   113   0.168                   insCTG   (+L)           C   G→T    1116   R46L   113   0.022           D   C→T    1120   S47S   113   0.016           E   C→T    1137   A53V   113   0.124               2   F   T→C    4824   Intronic   100   0.040               3   G   G→A    7464   Intronic   25   0.016               4   H   G→C   13327   Intronic   100   0.548           I   G→C   13349   Intronic   100   0.547           J   G→A   13406   Intronic   100   0.063           K   G→A   13559   Intronic   100   0.052           L   C→A   13626   Intronic   100   0.076           M   A→G   13632   Intronic   100   0.382               5   N   G→A   13753   Intronic   23   0.020           O   C→T   13781   Intronic   23   0.280           P   A→G   13932   Intronic   23   0.240           Q   A→C   13993   Intronic   23   0.170               8   R   T→C   19444   Intronic   20   0.175               9   S   T→C   19576   Intronic   113   0.137           T   G→A   19657   V460V   113   0.128           U   A→G   19697   I474V   113   0.141               10   V   C→T   20845   Intronic   24   0.040           W   A→G   20846   Intronic   24   0.146               11   X   A→G   22769   Intronic   20   0.030               12   Y   A→G   24633   E670G   79   0.082                    
         [0197]    [0197]                                                                                                       TABLE 3                           Haplotypes of affected subjects from the three ADH families                Markers in 1p32 region                PCSK9                    Exon 1   Exon 4   Exon 9                Family   Mutation   D1S417   Polym. B   Polym. H   Polym. I   Polym. M   Polym. U   D1S200   D1S2742               HC92   S127R   2   No   G   G   A   A   3   6       HC2   S127R   2   No   G   G   A   A   3   6       HC60   F216L   4   No   C   C   G   G   —   3                    
         [0198]    Marker haplotypes surrounding the mutations of PCSK9: markers are given in physical order from telomere (left) to centromere (right). A common disease haplotype segregates with the S127R mutation in both the HC92 and the HC2 family.  
                                                                   TABLE 4                           Mutations identified in the PCSK9 gene                Nucleotidic   Position   Amino acid               Exon   Variation   SEQ ID N o 1   variation   Effect                    1   +CTA    314-316   Insert L23                       1   +CTACTA    314-319   Insert LL23, 24               1   CAG→GAG    335-337   Q31N               1   TGC→GCA    443-445   C67A               2   ACC→GCC    473-475   T77A               2   TAC→GCC    476-478   Y78A   Inactive zymogen               2   GTG→GCG    479-481   V79A               2   GTG→GCG    482-484   V80A   Inactive zymogen               2   GTG→ATT    482-484   V80I               2   GTG→GCG    482-484   V80A               2   GTG→TTG    482-484   V80L   Inactive zymogen               2   GTG→GCG    485-487   V81A               2   CTG→GCG    488-490   L82A   Inactive zymogen               2   CTG→GTG    488-490   L82V   Partially active enzyme               2   CTG→CCG    488-490   L82P   Inactive zymogen               2   GTGGTGGTG→    485-490   V79R &amp; V80R   Inactive zymogen           CGGCGGCTG       &amp; V81L               2   GTG→ATT &amp;    479-481 &amp;   V79I &amp; K83M   Increased zymogen           AAG→ATG    491-493       activation               2   GAG→GCG    494-496   E84A   None               2   GAG→GCG    497-499   E85A               2   ACC→GCC    500-502   T86A               2   CAC→GCC    503-505   H87A               2   CTC→GCC    506-508   L88A               2   GCTGCC→CGTAGA    548-553   A102R &amp;   Inactive zymogen                   A103R               2   ACC→ATC    569-571   T109I   Increased zymogen                       activation               2   GTG→ATT &amp;    479-481 &amp;   V79I &amp; K83M   Inactive zymogen           AAG→ATG &amp;    491-493 &amp;   &amp;           CTG→CCG    578-580   L112P               2   ATG→GCG    620-622   M126A   Decreased zymogen                       activation               2   AGT→CGT    623-625   S127R   Decreased zymogen                       activation               2   AGT→GCT    623-625   S127A   Decreased zymogen                       activation               2   AGT→CCT    623-625   S127P   None               3   CCC→TAC    656-658   P138Y   Decreased zymogen                       activation               3   ATC→CCC    671-673   I143P   Inactive zymogen               4   TTC→CTC    890-892   F216L               4   AGA→AGT    896-898   R218S               5   CAT→GCT    920-922   H226A   Inactive zymogen               5   CGC→TGG    953-955   R237W               6   AAC→GCA   1193-1195   N317A   Inactive zymogen               10   AAC→GCG/GCA/GCC/   1841-1843   N533A           GCT               10   AAC→CAG   1841-1843   N533Q   Loss of N-glycosylation               11   AAC→CAG   2000-2002   N586Q               12   AAC→CAG   2198-2200   N652Q                  
 
         [0199]    [0199] 
     
       
       
         1 
         
           
             3  
           
           
             1  
             3617  
             DNA  
             Homo sapiens  
             
               CDS  
               (245)..(2320)  
                 
             
           
            1 

cccacgcgtc cggcctggag gagtgagcca ggcagtgaga ctggctcggg cgggccggga     60 

cgcgtcgttg cagcagcggc tcccagctcc cagccaggat tccgcgcgcc ccttcacgcg    120 

ccctgctcct gaacttcagc tcctgcacag tcctccccac cgcaaggctc aaggcgccgc    180 

cggcgtggac cgcgcacggc ctctaggtct cctcgccagg acagcaacct ctcccctggc    240 

cctc atg ggc acc gtc agc tcc agg cgg tcc tgg tgg ccg ctg cca ctg     289 
     Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu 
     1               5                   10                  15 

ctg ctg ctg ctg ctg ctg ctc ctg ggt ccc gcg ggc gcc cgt gcg cag      337 
Leu Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln 
                20                  25                  30 

gag gac gag gac ggc gac tac gag gag ctg gtg cta gcc ttg cgt tcc      385 
Glu Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser 
            35                  40                  45 

gag gag gac ggc ctg gcc gaa gca ccc gag cac gga acc aca gcc acc      433 
Glu Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr 
        50                  55                  60 

ttc cac cgc tgc gcc aag gat ccg tgg agg ttg cct ggc acc tac gtg      481 
Phe His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val 
    65                  70                  75 

gtg gtg ctg aag gag gag acc cac ctc tcg cag tca gag cgc act gcc      529 
Val Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala 
80                  85                  90                  95 

cgc cgc ctg cag gcc cag gct gcc cgc cgg gga tac ctc acc aag atc      577 
Arg Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile 
                100                 105                 110 

ctg cat gtc ttc cat ggc ctt ctt cct ggc ttc ctg gtg aag atg agt      625 
Leu His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Ser 
            115                 120                 125 

ggc gac ctg ctg gag ctg gcc ttg aag ttg ccc cat gtc gac tac atc      673 
Gly Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile 
        130                 135                 140 

gag gag gac tcc tct gtc ttt gcc cag agc atc ccg tgg aac ctg gag      721 
Glu Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu 
    145                 150                 155 

cgg att acc cct cca cgg tac cgg gcg gat gaa tac cag ccc ccc gac      769 
Arg Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp 
160                 165                 170                 175 

gga ggc agc ctg gtg gag gtg tat ctc cta gac acc agc ata cag agt      817 
Gly Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser 
                180                 185                 190 

gac cac cgg gaa atc gag ggc agg gtc atg gtc acc gac ttc gag aat      865 
Asp His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn 
            195                 200                 205 

gtg ccc gag gag gac ggg acc cgc ttc cac aga cag gcc agc aag tgt      913 
Val Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys 
        210                 215                 220 

gac agt cat ggc acc cac ctg gca ggg gtg gtc agc ggc cgg gat gcc      961 
Asp Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala 
    225                 230                 235 

ggc gtg gcc aag ggt gcc agc atg cgc agc ctg cgc gtg ctc aac tgc     1009 
Gly Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys 
240                 245                 250                 255 

caa ggg aag ggc acg gtt agc ggc acc ctc ata ggc ctg gag ttt att     1057 
Gln Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile 
                260                 265                 270 

cgg aaa agc cag ctg gtc cag cct gtg ggg cca ctg gtg gtg ctg ctg     1105 
Arg Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu 
            275                 280                 285 

ccc ctg gcg ggt ggg tac agc cgc gtc ctc aac gcc gcc tgc cag cgc     1153 
Pro Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg 
        290                 295                 300 

ctg gcg agg gct ggg gtc gtg ctg gtc acc gct gcc ggc aac ttc cgg     1201 
Leu Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg 
    305                 310                 315 

gac gat gcc tgc ctc tac tcc cca gcc tca gct ccc gag gtc atc aca     1249 
Asp Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr 
320                 325                 330                 335 

gtt ggg gcc acc aat gcc cag gac cag ccg gtg acc ctg ggg act ttg     1297 
Val Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu 
                340                 345                 350 

ggg acc aac ttt ggc cgc tgt gtg gac ctc ttt gcc cca ggg gag gac     1345 
Gly Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp 
            355                 360                 365 

atc att ggt gcc tcc agc gac tgc agc acc tgc ttt gtg tca cag agt     1393 
Ile Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln Ser 
        370                 375                 380 

ggg aca tca cag gct gct gcc cac gtg gct ggc att gca gcc atg atg     1441 
Gly Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met 
    385                 390                 395 

ctg tct gcc gag ccg gag ctc acc ctg gcc gag ttg agg cag aga ctg     1489 
Leu Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu 
400                 405                 410                 415 

atc cac ttc tct gcc aaa gat gtc atc aat gag gcc tgg ttc cct gag     1537 
Ile His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu 
                420                 425                 430 

gac cag cgg gta ctg acc ccc aac ctg gtg gcc gcc ctg ccc ccc agc     1585 
Asp Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser 
            435                 440                 445 

acc cat ggg gca ggt tgg cag ctg ttt tgc agg act gtg tgg tca gca     1633 
Thr His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala 
        450                 455                 460 

cac tcg ggg cct aca cgg atg gcc aca gcc atc gcc cgc tgc gcc cca     1681 
His Ser Gly Pro Thr Arg Met Ala Thr Ala Ile Ala Arg Cys Ala Pro 
    465                 470                 475 

gat gag gag ctg ctg agc tgc tcc agt ttc tcc agg agt ggg aag cgg     1729 
Asp Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg 
480                 485                 490                 495 

cgg ggc gag cgc atg gag gcc caa ggg ggc aag ctg gtc tgc cgg gcc     1777 
Arg Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala 
                500                 505                 510 

cac aac gct ttt ggg ggt gag ggt gtc tac gcc att gcc agg tgc tgc     1825 
His Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys 
            515                 520                 525 

ctg cta ccc cag gcc aac tgc agc gtc cac aca gct cca cca gct gag     1873 
Leu Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu 
        530                 535                 540 

gcc agc atg ggg acc cgt gtc cac tgc cac caa cag ggc cac gtc ctc     1921 
Ala Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu 
    545                 550                 555 

aca ggc tgc agc tcc cac tgg gag gtg gag gac ctt ggc acc cac aag     1969 
Thr Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys 
560                 565                 570                 575 

ccg cct gtg ctg agg cca cga ggt cag ccc aac cag tgc gtg ggc cac     2017 
Pro Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His 
                580                 585                 590 

agg gag gcc agc atc cac gct tcc tgc tgc cat gcc cca ggt ctg gaa     2065 
Arg Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu Glu 
            595                 600                 605 

tgc aaa gtc aag gag cat gga atc ccg gcc cct cag gag cag gtg acc     2113 
Cys Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu Gln Val Thr 
        610                 615                 620 

gtg gcc tgc gag gag ggc tgg acc ctg act ggc tgc agt gcc ctc cct     2161 
Val Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro 
    625                 630                 635 

ggg acc tcc cac gtc ctg ggg gcc tac gcc gta gac aac acg tgt gta     2209 
Gly Thr Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val 
640                 645                 650                 655 

gtc agg agc cgg gac gtc agc act aca ggc agc acc agc gaa gag gcc     2257 
Val Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Glu Ala 
                660                 665                 670 

gtg aca gcc gtt gcc atc tgc tgc cgg agc cgg cac ctg gcg cag gcc     2305 
Val Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala 
            675                 680                 685 

tcc cag gag ctc cag tgacagcccc atcccaggat gggtgtctgg ggagggtcaa     2360 
Ser Gln Glu Leu Gln 
        690 

gggctggggc tgagctttaa aatggttccg acttgtccct ctctcagccc tccatggcct   2420 

ggcacgaggg gatggggatg cttccgcctt tccggggctg ctggcctggc ccttgagtgg   2480 

ggcagcctcc ttgcctggaa ctcactcact ctgggtgcct cctccccagg tggaggtgcc   2540 

aggaagctcc ctccctcact gtggggcatt tcaccattca aacaggtcga gctgtgctcg   2600 

ggtgctgcca gctgctccca atgtgccgat gtccgtgggc agaatgactt ttattgagct   2660 

cttgttccgt gccaggcatt caatcctcag gtctccacca aggaggcagg attcttccca   2720 

tggatagggg agggggcggt aggggctgca gggacaaaca tcgttggggg gtgagtgtga   2780 

aaggtgctga tggccctcat ctccagctaa ctgtggagaa gcccctgggg gctccctgat   2840 

taatggaggc ttagctttct ggatggcatc tagccagagg ctggagacag gtgtgcccct   2900 

ggtggtcaca ggctgtgcct tggtttcctg agccaccttt actctgctct atgccaggct   2960 

gtgctagcaa cacccaaagg tggcctgcgg ggagccatca cctaggactg actcggcagt   3020 

gtgcagtggt gcatgcactg tctcagccaa cccgctccac tacccggcag ggtacacatt   3080 

cgcaccccta cttcacagag gaagaaacct ggaaccagag ggggcgtgcc tgccaagctc   3140 

acacagcagg aactgagcca gaaacgcaga ttgggctggc tctgaagcca agcctcttct   3200 

tacttcaccc ggctgggctc ctcattttta cgggtaacag tgaggctggg aaggggaaca   3260 

cagaccagga agctcggtga gtgatggcag aacgatgcct gcaggcatgg aactttttcc   3320 

gttatcaccc aggcctgatt cactggcctg gcggagatgc ttctaaggca tggtcggggg   3380 

agagggccaa caactgtccc tccttgagca ccagccccac ccaagcaagc agacatttat   3440 

cttttgggtc tgtcctctct gttgcctttt tacagccaac ttttctagac ctgttttgct   3500 

tttgtaactt gaagatattt attctgggtt ttgtagcatt tttattaata tggtgacttt   3560 

ttaaaataaa aacaaacaaa cgttgtccta aaaaaaaaaa aaaaaawaaa aaaaaaa      3617 

 
           
             2  
             692  
             PRT  
             Homo sapiens  
             
               ACT_SITE  
               (186)..(186)  
               Asp  
             
           
            2 

Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu 
1               5                   10                  15 

Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu 
            20                  25                  30 

Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Xaa Ser Glu 
        35                  40                  45 

Glu Asp Gly Leu Xaa Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe 
    50                  55                  60 

His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val 
65                  70                  75                  80 

Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg 
                85                  90                  95 

Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu 
            100                 105                 110 

His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Xaa Gly 
        115                 120                 125 

Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu 
    130                 135                 140 

Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg 
145                 150                 155                 160 

Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly 
                165                 170                 175 

Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp 
            180                 185                 190 

His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val 
        195                 200                 205 

Pro Glu Glu Asp Gly Thr Arg Xaa His Arg Gln Ala Ser Lys Cys Asp 
    210                 215                 220 

Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly 
225                 230                 235                 240 

Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln 
                245                 250                 255 

Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg 
            260                 265                 270 

Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro 
        275                 280                 285 

Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu 
    290                 295                 300 

Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp 
305                 310                 315                 320 

Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val 
                325                 330                 335 

Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly 
            340                 345                 350 

Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile 
        355                 360                 365 

Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln Ser Gly 
    370                 375                 380 

Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu 
385                 390                 395                 400 

Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile 
                405                 410                 415 

His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp 
            420                 425                 430 

Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr 
        435                 440                 445 

His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His 
    450                 455                 460 

Ser Gly Pro Thr Arg Met Ala Thr Ala Xaa Ala Arg Cys Ala Pro Asp 
465                 470                 475                 480 

Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg 
                485                 490                 495 

Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala His 
            500                 505                 510 

Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu 
        515                 520                 525 

Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala 
    530                 535                 540 

Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr 
545                 550                 555                 560 

Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro 
                565                 570                 575 

Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg 
            580                 585                 590 

Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys 
        595                 600                 605 

Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu Gln Val Thr Val 
    610                 615                 620 

Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly 
625                 630                 635                 640 

Thr Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val 
                645                 650                 655 

Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Xaa Ala Val 
            660                 665                 670 

Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser 
        675                 680                 685 

Gln Glu Leu Gln 
    690 

 
           
             3  
             26220  
             DNA  
             Homo sapiens  
             
               exon  
               (980)..(1186)  
               exon 1  
             
           
            3 

ctggatgctt gtccagttga tttcttgaac atggtgtgta aaaggaatct ttgcaaattg     60 

aatcttctgg aaagctgagc ttgtgcctac catagaattc tgaatgtacc tatatgacat    120 

ctttgcaaac ttaaaacctg aatctttgta gtataaatcc cttgaaatgc atgtaggctg    180 

gacatcaaaa gcaagcaatc tcttcaagga gcagctagtt ggtaaggtca gtgtgcaggg    240 

tgcataaagg gcagaggccg gagggggtcc aggctaagtt tagaaggctg ccaggttaag    300 

gccagtggaa agaattcggt gggcagcgag gagtccacag taggattgat tcagaagtct    360 

cactggtcag caggagacaa ggtggaccca ggaaacactg aaaaggtggg cccggcagaa    420 

cttggagtct ggcatcccac gcagggtgag aggcgggaga ggaggagccc ctagggcgcc    480 

ggcctgcctt ccagcccagt taggatttgg gagttttttc ttccctctgc gcgtaatctg    540 

acgctgtttg gggagggcga ggccgaaacc tgatcctcca gtccgggggt tccgttaatg    600 

tttaatcaga taggatcgtc cgatggggct ctggtggcgt gatctgcgcg ccccaggcgt    660 

caagcaccca caccctagaa ggtttccgca gcgacgtcga ggcgctcatg gttgcaggcg    720 

ggcgccgccg ttcagttcag ggtctgagcc tggaggagtg agccaggcag tgagactggc    780 

tcgggcgggc cgggacgcgt cgttgcagca gcggctccca gctcccagcc aggattccgc    840 

gcgccccttc acgcgccctg ctcctgaact tcagctcctg cacagtcctc cccaccgcaa    900 

ggctcaaggc gccgccggcg tggaccgcgc acggcctcta ggtctcctcg ccaggacagc    960 

aacctctccc ctggccctc atg ggc acc gtc agc tcc agg cgg tcc tgg tgg    1012 
                     Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp 
                     1               5                   10 

ccg ctg cca ctg ctg ctg ctg ctg ctg ctg ctc ctg ggt ccc gcg ggc     1060 
Pro Leu Pro Leu Leu Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly 
            15                  20                  25 

gcc cgt gcg cag gag gac gag gac ggc gac tac gag gag ctg gtg cta     1108 
Ala Arg Ala Gln Glu Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu 
        30                  35                  40 

gcc ttg cgt tcc gag gag gac ggc ctg gcc gaa gca ccc gag cac gga     1156 
Ala Leu Arg Ser Glu Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly 
    45                  50                  55 

acc aca gcc acc ttc cac cgc tgc gcc aag gtgcgggtgt aggggtggga       1206 
Thr Thr Ala Thr Phe His Arg Cys Ala Lys 
60                  65 

ggccggggcg aacccgcagc cgggacggtg cggtgctgtt tcctctcggg cctcagtttc   1266 

cccccatgta agagaggaag tggagtgcag gtcgccgagg gctcttcgct tggcacgatc   1326 

ttgaggactg caggcaaggc ggcgggggag gacgggtagt ggggagcacg gtggagagcg   1386 

gggacggccg gctctttggg gacttgctgg ggcgtgcggc tgcgctattc agtgggaagg   1446 

ttcgcggggt tgggagaccc ggaggccgag gaagggcgag cagagcactg ccaggatatc   1506 

ctgcccagat ttcccagttt ctgcctcgcc gcggcacagg tgggtgaagg agtgaatgcc   1566 

tggaacgtac tgggaactgc accaggcaca gagaaagcgg gcttgccatt atagtgggtt   1626 

ccgatttggt ttggaaaaca tgggcagcgg agggtggagg gcctggagag aaggccctac   1686 

ccgagacagg ggcggggtgg gaaggacggc agatgctggg agcacgaggc aatttcttta   1746 

tgacacagaa ctcatgctct agtattccat ctgtttcagc cgaagaaaag aaccagctga   1806 

aggggcaggg gagaaggggc ggaggtattc tcgaggccca ttggcgtcct ttaggactca   1866 

ggcagggaag ggcccttggt gctctggagc cggaggtggt gcgcctggta ctgggacccc   1926 

ggagctgagc ccggcgcctc agcccacctg gctgtctgcc gaccgtgtgc ggggcgagtt   1986 

tgctcaacaa ctctgccagc ttctggccct caggctgtgg gaagcttctt cccggggcga   2046 

gaccactagc tttttctaag tattaccagc ccaggacttg gctgaggttc tgtgtccccc   2106 

agcttggagt cagatgtggg gttgaatctt ggcttcctct cactagctgt ggtgcttgac   2166 

aagtcactta tccttgagcc tccattgcct aatctttaaa agggaggtga caatcgtccc   2226 

tacggctcag tggcagcaga tggggagatg aagggaaagt tctgttgacc atgagtgaac   2286 

ttacaatgca agccccgggg ggatcacttg cagttttgtc cctgtctgca gtgtgacctg   2346 

ttggtgacat tgtctttgct ccaaaccaca gctcctgggg cagaggggaa aattctgcca   2406 

ctcacagctg cctgcccacg cttctgtctg agtgtgctgg gtggcaggat ggcaagtcct   2466 

tactcagctc agtatagccc tcttccttgt tccctgagcc tttgactttc tcgagggatg   2526 

ttgtggggtt gtggccagga taagaaaggg catttcaagt taccactgct ccaaaacaac   2586 

tgttctggaa atagtgagta ccccatcctg agaggtgagt aagcagaggc tgtatgacca   2646 

cctgaaccaa gcccttgagg atgtttcttc tctggtggaa gtttggaaca ggagcctcct   2706 

caagttcatt tattcattca ttcaatggtt attttgtggg aatcgaattt agaatgaaaa   2766 

tattttttgg caagcagaaa ataattttta gaccaatcct tttcttttag tcatgagaaa   2826 

ctgaggccca gagagaggag gtcaccccag gtgcattaga actgggtttc cagaactgac   2886 

actccactgc acagagtact ctcccaattc attcaatttt tatttagcgg aaggcatttt   2946 

cagatgggtc tttgaagcat tagtaggagt tcagcgatga tggtgtcatg agaattttat   3006 

tctaggatta ggaggtacca tgaacaaaga tacagagctg ggaaaaccag aggtggaaga   3066 

taaggagcac atgtccacag ttctttttct tttttttttg agatggagtt tcgctcttgt   3126 

tgcccaggct ggagtgcaat ggtgcagtct cagctcactg caacatctgt ctcccgggtt   3186 

caagtggttc tcctgcctca gcctcccaag aagctgggat tacaggtacc tgccaccacg   3246 

cccggctaat ttttgtattt ttagtagaga aggggtttca ccacgttggc caggctagtc   3306 

gcaaactcct gacctcctca gtggatccga ggaggtgatc ctcccgcctc agcctcccaa   3366 

agtgctcgaa ttacaggtgt gagccaccac gcctggcctc cacagttctt tatccaccgt   3426 

ctgaaatgta aaatgttacg aaaaccaaaa gttttttttg tgatttattt gatggtagca   3486 

cctgacgtga actgacatga gattattttt aatttagttg tgtgaatatg catattcata   3546 

tattttgctg catagattac agtatgcagc tccagattct tccaagcaga ctctgattgc   3606 

ccattactgc ctttctaaaa tccaaacaag ttctgaggtt caaaaccgat ttggccctaa   3666 

ggctttgggt aaagggggtg gactctgttc tactctgact ggagtccaag atgcatatat   3726 

acagagatat gggtgatggg gctgcaaggt aggttgaggt aggggccaag gaggagcatg   3786 

gagtttggac ttgattcatg aggctgtggg gagccagtga aggttcttaa gcaggtatgt   3846 

ctgcctgaga gcagttggag cagacaagag ctaaaaacca aacaaatcac catagatagt   3906 

ggctgctata atttgtttgt cccctccaaa tctcatgtgg aaatttggtc ctcagtgttg   3966 

gaagtggggc ctaatgggag gtgtttgggt catgggggag gaacccctgt gaaaggcttg   4026 

gtgccgtcct tgtgataatg agtaagttct cccgctatga tttcccttga aggctgatta   4086 

ttaaaaagag cttggcacct ccctctcttc tctcttgctt cttctcttgc catgtgattg   4146 

atctctgcac atgtaggctc cccttcacct tctgccatca gtgaaagcag cttaaggccc   4206 

tcaccagaag cagatgctgg tgccatgctt cctggagagc ttgcagaatc atgagctgaa   4266 

taaatccctt ttccttgtaa attactcacc ttcaggtatt cctttatata gcaacacaaa   4326 

aggactaaga cagtggcctt gacttttctc tctctttaag aagtgttgcc tttgctcact   4386 

tagtcatccc ttctgcctgc atttgtagag catctggatg ggagatttat ataaccgtca   4446 

ctcttgactt tcccagcagg cctatgtcat aggtactgtg gtctctacaa tacagcagag   4506 

gtatctgagg ctccgagagg ttgagtgact tgctcatggc tgcacaacca gtaaatattg   4566 

gagctggaat tcaggtccac ggtttcctgg ctccaaagcc catgattttt tccctcaatt   4626 

tattctgact ggggcatggg ggagggggtg gcctttgggc agggccacca ggagcgacca   4686 

ggcccgtaga gagctgggtg caggtacaga ggaaaacctg ttgtcgagtg tggcccgtag   4746 

ttcccatttt tgcctgaatg gcacatttga aagtgttata taaccatgtg aataataata   4806 

gttggcctat atgagttttt taatttgctt tttggtccgc atttggtaac ttctttatca   4866 

tctactatac tctgttgtgt ctcttttgtt gtaatttgta agtaggggtg agataaagta   4926 

cacctagggt ttgctgggtt tcttccatgt catcatgttc ctccttgcat ggggccag     4984 

gat ccg tgg agg ttg cct ggc acc tac gtg gtg gtg ctg aag gag gag     5032 
Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val Val Leu Lys Glu Glu 
70                  75                  80                  85 

acc cac ctc tcg cag tca gag cgc act gcc cgc cgc ctg cag gcc cag     5080 
Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg Arg Leu Gln Ala Gln 
                90                  95                  100 

gct gcc cgc cgg gga tac ctc acc aag atc ctg cat gtc ttc cat ggc     5128 
Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu His Val Phe His Gly 
            105                 110                 115 

ctt ctt cct ggc ttc ctg gtg aag atg agt ggc gac ctg ctg gag ctg     5176 
Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly Asp Leu Leu Glu Leu 
        120                 125                 130 

gtgagccacc ctttttggga atggcacttc ctgatagggc tgggccactg catatacact   5236 

ggggactgtg cttagtaggc ccattgctga aaatcagaag gggacagcaa gtatgtattg   5296 

agcacttatc gggtaccaag cacagtaact actggctttc tgtatagaat tccctttaag   5356 

cctggccatg ccccagtggt acgtctatct tcatttgaaa gacgaggaga ctgaagttca   5416 

gaggggacca cacagacagc taggggtaga gcctggatca aacccattgg tctgcctgcc   5476 

agccattctt gtgccaatgc atctgctgcc tacggaaacc tgtagggaca aggccctggg   5536 

atgttcagtg gagcctgagt cattttataa aaaagcatga ctctagggtc caaaattcct   5596 

ttgaagctgt tgctatccag agtgaagtcc cttctttagg acagggtggc cctcctccct   5656 

cctggatgtc acatcttcgg tggaggggca gaaaggggac tgggtattct cctcaccctg   5716 

gccctagtgc ttcaaatctt aaaaaaacgt ttttatttgt gcttctgcac caccttctag   5776 

cccacctcgt ttcctggcct ctaacttgat gagagcgtgt gtcattttca cactgattct   5836 

ccacatggca ggcggtgctt cttagcctcc tgcagacagt gaggccccac ggtcttgtcc   5896 

aaggtcacac agcgtgtaat gggcagggtc agagtctgga gtctggacct gggtctccta   5956 

gctgcactgc actgctgccc catgggttaa tcagctcagc ataccgtggc tgaacagcta   6016 

cctcatacca aggcctgtgg cgccatgaca gggattgaca gggtccctgc cttggaaacc   6076 

cgtagtctaa gtagaggaga ctgacaagtc aatgccttcc atcagtctgc tcaacacacg   6136 

tttaccaagt gcctactgtg tgctgcagag gcgaagatga cacagctcag gcctttccct   6196 

tgagcttaca gttcaggagg agagactgac cagtgactgc cagtacagtt gactatggga   6256 

caatgtgctc agccttgggg agagacgaag aaggtacccg tatagcacca gatgacaggc   6316 

acgagcccca caggccaggg cagctgctca gaggagagta ggccaagcag aaggcaaaca   6376 

gaaggctgca ggcatttgcc atcgagagct ggacttcaaa ctgggcatca taccagcctg   6436 

ggttcgagtc ctgcccagcc ccttattggc tgtctaaccc tgagcaaatc ccttcacctc   6496 

tctgagcctc attcctctat ctgtaaacca gttataataa ttggaacatt catttaagga   6556 

ctaaatgagg tcgtgaagca ttcagcagat gctaggtacg gaaactcgct gaagtggggg   6616 

caggttaaga agcctctggg gatacgaagg catccaggga ctagttgtgg caggaggctg   6676 

ttaccactta ggtctgaagg gtaaggagag ggaatagctt tccctctgcc cagttggagc   6736 

cggtggcatg gaggagaggc tgcctgtggg gaatcacccg agggttcacc gctgccatgc   6796 

gcagggagtc aggaggtagg gagggagtgg ggcagatgca caccattttt tttttttttt   6856 

gagactctgt tgcccagact ggagtgcagt ggtgccatat ctgcacctct gcctcccggg   6916 

ttcaagctca ctgcaacctc tgcctcccgg gttcaagcga ttctcctgcc tcagcctccc   6976 

gagtagctgg gactacaggt gtgtgccacc atgcctggct aatttttgta tttttaatag   7036 

agatggggtt tcaccatgtt ggccaggctg gtctcgaact ctcgacctca ggtgatcccc   7096 

cacctcggcc tcccaaagtg ctgggattac aggcgtgagt caccgctccc agctgctgat   7156 

gcactcttgt ccttctaact cctgctagtg cctcccattg gctgagccca actggaagct   7216 

ttgcaaggga gctggtgctg cagtttgcac tgagcaggct ggagaaggct ggagaataga   7276 

ctaggggaca aaccgaattg ccagtgctgt tatgtcatga tttaggcatg gagtccaggg   7336 

cctgagcttc actccatgtc catcctgccc agagccttgg cacagcctgg ctcccagaca   7396 

agatgtcaag ttcagaatcc ttcctaaaag gaatcctcta tgccagaccg tgttgcaggg   7456 

atatgggggt gctgggctcc cagcctgatc aaggagcgag aaaactcagg ctcctagtct   7516 

gtcctccggg gcactagcag ggacaaggtg ggaggctgct gggctgggat gtggggacag   7576 

gtttgatcag gtaaggccag gctgtggctg tgtttgctgc tgtccaaatg gcttaagcag   7636 

agtcccccgg cctctctggc ttctgcag gcc ttg aag ttg ccc cat gtc gac      7688 
                               Ala Leu Lys Leu Pro His Val Asp 
                                   135                 140 

tac atc gag gag gac tcc tct gtc ttt gcc cag agc atc ccg tgg aac     7736 
Tyr Ile Glu Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn 
            145                 150                 155 

ctg gag cgg att acc cct cca cgg tac cgg gcg gat gaa tac cag ccc     7784 
Leu Glu Arg Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro 
        160                 165                 170 

ccc g gtaagacccc catctgtgcc ctgccccacc ccatctgagc tgaatccatt        7838 
Pro 

tgctctgccc tggcctggcc tccctgctgg tggtttccac ttctcggggg gctttgggac   7898 

tcagcacctc cactgacccc tttttttctg tcccatcccc atcccctgca gcccccactg   7958 

cctgccttcc tgttgcccca caaatgcaaa agtcttgcct taaatgatcc tcttttcctc   8018 

cttttctctt gttttccttt tctcaccatt tggaatggcc cagcaggctg cacttacctt   8078 

ggaaggaggg ttcatctgat ggtgactcta cctagggccc ccaggcctct ataactccca   8138 

gtgccctgca gactggacca gatcctttaa tgggatagac acaaccctgt ctgggatgcc   8198 

tctgcctacc ttcctgtttt gctgctccac ctgcctccag ctccgtttgg cttcctgggg   8258 

ctccctgcct gggccacttt gtgtcttccc tctaggcctt tctttccact gttccctctg   8318 

cctggtgtgg cctggctatg gaagggaggg agcaggagcg gccatggaaa acggtctgca   8378 

ttctagcagg gacttgcagg tggcaattca gtcggggaag actctagatg cacctggcct   8438 

gaggagagaa tgaagggttc tagttggact gtgttaagtt tgaggtgccc atggtgtgag   8498 

gtctggagct cagcgcagag atgatgcaat gtggtgggtc catgcaacat ggtgccagga   8558 

cgcagagctt ggggtgaact cagctttcac cccttaccgg ttctcgtggg atcttgggaa   8618 

gccactttct tctatgagct ttgtcgttct tgtctgtaaa atgggcacat aaccctgtcc   8678 

ctgtccttct cacaggttgc tgtgagactc caatgagttg aaggatgtgc agatgctttt   8738 

ggaagtgaaa agttgggggg ctactgtgtg actttgcata cacccaaact gtgtgacctt   8798 

gcatatgtct gagttgctgc cattgcaaca gatcagagct ggtgggctgg gtgtggagaa   8858 

agggtttgtg tgggggacat cctctggcaa gggtggcagc agcagaagtg aggggcctgg   8918 

tcggtcatgt gtgctgaccc ggcctgggca gcctgtggcc agggagagga cagctcctct   8978 

gtaggaagag cgtgttcctt tccaaccagg tgagacctct tcagtggagc cctggagccc   9038 

cctgtactcc acatcagtgc ctcagggacc tcccggagca ggctaatatc agagaccaag   9098 

agggacactg gcagaggatc acagagaccc cagtccaggc agggactgag aagatcttgc   9158 

cccctaagtt agtttcctag cactgctgtg acaaaatacc accccctcgg ttggaacaag   9218 

ttgattctct gcagtcctgg aggccagaag cctgaatcag tgtcggcagg accactttct   9278 

cccggggggc tccagggaga agcttctctt gcctcttccg tgtcccaaca gcggcagcac   9338 

accaatccca gcctctgtct tcacacagcc ttctctgtgt ctctctcctc ttcattgtct   9398 

cataaggaca cttgtcattg gatttagggc ccactggatc ctccaggatg atctcatgtg   9458 

gggaacctta accacatctg caaggaccct ttttccaaat aaggtcacag ccacagtttg   9518 

tgggggttag gatgtgagtg tatctctttg gcagccactg ttccctcctc tcccttgggc   9578 

cagaagcaga cgtggggccc tttcttcccc ataggatgcc catggattgc cccccttccc   9638 

gcttcccccg agcgtctgtg ggaggtggca ggaatggcag gcaggtgtgt ggaacccctt   9698 

ctggagtcat atcaagggct tggctggagg aagtcctcct ggagctgttg ggctggcatg   9758 

gggcaggctg gctgggccca gcagcagctt cttcattcat ggggaggcca caagcatggg   9818 

ccctagagct ggctgccgcc ctcaaaccca gaccctgcac tcttaactgt gtgaccttgc   9878 

atacgtcact caccctctct gatcttcagg ttcctctgca aaagggaggt aatgataacc   9938 

ctcactctgg ggggctgttt ggagggttaa atcagttatt gctgtagcat gcatttctct   9998 

gtcaggtatt gagtgaggtg ctgtgatttt agccctgcat ttttcttttc ttaccattca  10058 

ataataacgt tttgagcacc ctctgtgcgc caggcaccat attaggtgct ggggatacaa  10118 

atgtgaatga aatgaatgtg gtctctttcc ccaacagtgt atccagaaga ttaatccatt  10178 

ccttaaacaa atgctacttg acacagatta gttctggata ggctgagagc tctgaaggag  10238 

tgcaggcagc tgcgagcctg tgtatccagc agaaggatca ggaaaggatt cctggaggaa  10298 

gcgctgttct agccaagacc tacgggggca ttattaacca ggcaaagggg acggtgtcca  10358 

agcagtggaa tgaacgtgga ttgaagctgt gaggcaggag ggagtgtggc ctgtgcagaa  10418 

gggaccgagg ctggtgagac cagcagggcc tgggtggcct ccaggtcaga tgtgaaagga  10478 

agaacttggc cacagtctga gcttctcagg cgtatggcag ggctgcctgg tgagagggaa  10538 

tgagctccct gctctggagg tatgcaagca ggactgggct ctcacctgcc agaggccaca  10598 

gagctttcca gaggctggaa gaggccactc caaggcctct ttgcccctga gagtggtggc  10658 

tcttcttgag gccaccttgc cacgctgtca cagggaacta gcagcccctg cctcacccgg  10718 

gggtttggaa gatagaggga ggcctaggaa gggccctgtg tctcatccga gctgggcccc  10778 

tttccagcct ctcactggaa ggaagcccaa ggatgttcct gtgggggctt ttaccaggcc  10838 

cacctgccct ctgctggcca tgcttgcagc ctcctgaccc tgtcccagca ggacagtggg  10898 

ctggtgtgag cgggcaggaa ccgcctgcac ttagaaggtg tggggctgcc tccccgagct  10958 

tccatctgcc gctggggcca caccccaggc ccagggatgg gaccccatag tggtcacatc  11018 

atcttgcagc agaacccagg tacagctcct ggagcagatg gtggtcccaa gcacgggtgg  11078 

gaccagaaag gactctcacc tgggctaact cagctgcagc ctcagttccc tcctcacaca  11138 

cgaggaacat ggactggaag cctgcccagc aggccttctg ctcgatgtgc gttgtgtggc  11198 

ttacgtccag ggagggaagc agcctctgtg ctgtcttcta gataagcctg tattccccgg  11258 

gctgtctgcc aatgtatcca gttgtcccgt cagcctggaa gctctgaggg aaaaccttgg  11318 

gctgcttcct gagcacctgt atcccctgca gccagcccgg ggcctctgct aggagcagac  11378 

tgagcatggc ttatgggcct ggcaccatct ggcctctgcc caccttgctg gccttgtctt  11438 

gtgtctgccc cttcgacatt ccatagccca gctcaatatc tagtggttcc tctagggtgg  11498 

cgagcactgt ttggtctcca gatgtcttca ggtcggagct cacagcgctc tcagccaccc  11558 

cttcccagtg tagcaccggg cacatggtag atgcctattg atgagtgaaa gctcctaaca  11618 

cactcagaga gcaaggactc cgcctcatcc cacagcctgg gaggagaggc agactgccaa  11678 

ggacctgctc agcatgctac agaagaaacc aaagtgccca cgggactgat cagtggagct  11738 

tcctgccgag actggaggcc ttagggcagg gtagacagtg tgtgtgcagg ctggggactc  11798 

acagttcgga ctgtgcccag acctactagc atagtgggtg ggtgggagga tgcgggactg  11858 

ggggccgacc ttgcctgaaa ttcatgtggg atctcagagc agccactgaa ttgctctgta  11918 

gggggctaaa tagtggcccc cacagataca cacacccaga cagagcctgt gagccagacc  11978 

ttatttggag aaaaggtctt tgtagatgta attaagcatc tcaagatggc atcatctgga  12038 

ttatgcggtg ggctgtaagt cctgtgatgt gtctttatga gagaaaggca gagggagatt  12098 

tgacacacac aggaggggcc acgtggagac agaggtggag attggagaaa tgtggccaca  12158 

agccagggaa caccagcagc caccagaagc cggaagacgt gaggcagggt tcttcccaga  12218 

gccttcgctg ctgagtctgg gaatttgtta ccgaagccat aagaagtggg tacacgccct  12278 

gagcctccca cacttgctca cctgtcctga gatgagaatc tctactctgc agcatatttg  12338 

gaggatcact gcgggggcca cagaggtgct gttcagatgg cacttcagaa gactcaggag  12398 

accctggggc aggagcagtt tgactgacag cccagagggc tgccctctga ttccacctga  12458 

ggccctgctt ttcctggctg caggggttcc agggccaggc catttccgct ggcgcaggac  12518 

tctgctagca gcaacctgcc tgaagtcttc ctttggcctg gctgagagtt tctgagacct  12578 

gcgctggagc ggaggtgctt ccttccttgc ttcctttctt cctctctccc ttctccatcc  12638 

agcaggctgg acctgcctgg catctgtgag ctctccctac tttctcctat accctaacct  12698 

ttgtcctgca tgggcgactc ccccagtgag tctcttgcag cttttacccc agtgcctgct  12758 

tcttggagaa tccaaactga tccagttagg gatgataaag tgtagggtag gtgctcggtg  12818 

actgttttct ctgaggttgt gactcgtgtg aggcagaagc agtccccgtg agccctcctg  12878 

gtatcttgtg gagtggagaa cgcttggacc tggagccagg aggcccagac atacatcctg  12938 

tccgagctgc agcttcctgt ctctaaaatg agccggccag cgcaggtggc cagacatcac  12998 

tgttattctc ctttgagtct ttaaatcttg ttgtctttct tgcagactcg gtgagctgtg  13058 

aaaggctata ataggggctt tattttacac tttgatacta ttttttgaac attcatatta  13118 

ttgttagata ttgatattca tatgaaggag caggatgact tgggtccttc ttggcagtag  13178 

cattgccagc tgatggcctt ggacagttac ctgccctctc taggcctccc tttccttgtc  13238 

tatgaaatac attatagaat aggatgtagt gtgtgaggat tttttggagg ttaaacgagt  13298 

gaatatattt aaggcgcttt caccagtggc tgggatgtgc tctgtagttt gtgtgtgtta  13358 

actataaggt tgactttatg ctcattccct cctctcccac aaatgtcgcc ttggaaa     13415 

gac gga ggc agc ctg gtg gag gtg tat ctc cta gac acc agc ata cag    13463 
Asp Gly Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln 
175                 180                 185                 190 

agt gac cac cgg gaa atc gag ggc agg gtc atg gtc acc gac ttc gag    13511 
Ser Asp His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu 
                195                 200                 205 

aat gtg ccc gag gag gac ggg acc cgc ttc cac aga cag gtaagcacgg     13560 
Asn Val Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln 
            210                 215 

ccgtctgatg ggagggctgc ctctgcccat atccccatcc tggaggtggg tggggactgc  13620 

caccccagag cattgcagct gtactcctgg gttgcacccc cccagctgtc actgtcccct  13680 

ccctgccatc agttgtggga agggcgttca tccatccagc cacctgctga tttgttatag  13740 

ggtggagggg gggtctttct catgtggtcc ttgtgttcgt cgagcag gcc agc aag    13796 
                                                    Ala Ser Lys 
                                                    220 

tgt gac agt cat ggc acc cac ctg gca ggg gtg gtc agc ggc cgg gat    13844 
Cys Asp Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp 
        225                 230                 235 

gcc ggc gtg gcc aag ggt gcc agc atg cgc agc ctg cgc gtg ctc aac    13892 
Ala Gly Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn 
    240                 245                 250 

tgc caa ggg aag ggc acg gtt agc ggc acc ctc ata g gtaagtgatg       13939 
Cys Gln Gly Lys Gly Thr Val Ser Gly Thr Leu Ile 
255                 260                 265 

gccccagacg ctggtctctc tccatctgga cctggcctgg gaggtggctt gggatgggcc  13999 

cagggagagc taatgtctcc taaccaagaa tgctgtggca gcctctgccg cagagccaga  14059 

gaaccagagt gccaaggctg gcagggttcc cagtggccac gagtgcagat gaagaaaccc  14119 

aggccccaag agggtcatgc aggtagccca gggagttcag ccttgaccct gggtcaatga  14179 

cctttccaca gttccacact gctccccttt taaaatccgg tgatgtcttt atgtcttttg  14239 

ttatgttatc ttcaatgtgg agggactcga ggtgatctaa gcaaactttt tctatcttct  14299 

gcttgcatac ctctgagacc aggggactca ctcacttgca tgactgggcc ctgcaggtca  14359 

cactggccag gcagatgtgg tggaggaact ggcagaggac tttttctaga ctgtgactac  14419 

atttagtcca cccagcggcc cccctatgaa gtccagttga gaactaggac tctgggggcc  14479 

tgtggacaga gaagagggag ggttctctcc cttactgact tccttctgtg gccagacatt  14539 

gagcaaggcc tctgtacagc atgtcctggg gctggccttg ccgtagctgc taaatagttg  14599 

acgaaaccag tccagagagg ggaggtgact gccagggtca cacagctcaa gctggggaac  14659 

tcgctgggaa aactgtcagc tctgggcagc agcttgactt ccattgtaag ccccagcccc  14719 

cagggtcaaa cactggctct ggtgctggca gaggcagccc actagcctgt ttcaaaggct  14779 

gagaaggccc aggagtctgc cctgtgctcc accagttctg ccctgagact ttcctacaga  14839 

gtacaggttt tgatgttcag ttttaaaggc aagaatcaat aaccttctgc cccatcaggt  14899 

gaccccttgt gcctgtccca cccctttatt gactgacctc ggctcagtca ggtcagttcc  14959 

tgaaggtcag tgtgtggagg ggaggctgtt ctttcccaga aaggccttcc ccaggcctgg  15019 

tgctctggcc tctggaggac ttcctggaga agtcccttct ttggggtccc agtcagtgta  15079 

tgggaagccc ttattgcatg acctggcacg gggtaggggc tcaacagtca ctattgcctt  15139 

ccttgccact gccatttcct cctctgtaag caggtgattg tgtgtccagt ctgagcacag  15199 

agataagcac acagcaggtg cttaataact agcagctgta ggctgggcgc ggtggctcat  15259 

gcctgtaatc ccagcacttt gggaggccga ggtgggcaga tcacctgagg tcaggagttc  15319 

gagaccagcc tgttcaacat ggtgaaaccc cgtctctact aaaaatacaa aaattagcca  15379 

ggcatggtgg tgggtgtctg tatcccagct acttgggagg ctaaggcagg agaatcgctt  15439 

gaacccagga ggtggaggtt gcagtgagct gagatcgtgc cactgcaatc cagcctgagt  15499 

gatagagcga gattccatct caaaaataaa taagtaaata actagcagct gtaaatgtgg  15559 

ctgttgttct tcacctccac actcagtgcc actccactcc ctccctccgt ggtgtgaggg  15619 

gcctcactag ctgtctccta ggaggagcat ggctgtgaga ttccagctcc atccttgacc  15679 

acggctcctg gagacatctt agaggccagg atccagaagg ctcccacacc ccatttgaca  15739 

ggggagaagc tgtcagttcc aggtcccctt gcacatcagg gccagagctg cgttaggcct  15799 

ccagtctcca ggccactggg ccagagctca caggctggca gagggttaga actgttactg  15859 

gtggctgggt gcactggctc acgcctgtaa tcttagcact ttgggagggc aaggcgggag  15919 

gatcatgagg tcaggacatc gagaccatcc ttgctaacac ggtgaagccc cgtctctact  15979 

aaaactacaa aaaattagcc gggcgtggtg gcaggcgcct gtagtcccag ctactcagga  16039 

ggctgaggca ggagaatggc gtgaacccgg gaggcggagc ttgcagtgag ccgagattgc  16099 

gccactgcac tccagcctgg gcaatagagc gagactccgt ctggaaagaa aaaaaaaaaa  16159 

agagctgtta ctgttgacag tagcatgagg tagaccatgg cctgcaccaa aagggggagt  16219 

ggagtgccac tgaggccaga aggaaccaca ccctcaaggg tggggagtta tggtatgggg  16279 

ggtcctaggc atggagtctt ttaattcttt agacaatcct gggagcagct gtccctgttt  16339 

cacagagggc ggggccacac agctggtgag tgggcagcca agactctgtt caagtttgtg  16399 

tgggtccaac acttgcggcc acggtggagg ggcatctgag ccaggcctca gagagtggcg  16459 

gggggaagtt gggtggggaa gtgtgccctt ctcattcctc tgaggctcat cctcttggtg  16519 

cctctctttc atggaaaggg ataataaggt tattgtgagg atcccctgag ttcatatatt  16579 

cagacgctta gacagagcca ggcacagaga agggcccggg gttggctagt ttgattgctg  16639 

gtgtaattgc taatatcttc cagtttgtat tggtcaaggt tctgcagaga agcagaacca  16699 

gtaggaggta tatattaaga gtttcaagct catgtgaccg tgcgggctgg caagtctgaa  16759 

atccgcaggg caggccatgc aggctggcaa ttcctgcaga atttgatgtt gcaatactga  16819 

gtcctaaggc agtcctgggg cagaattcct tcttccctgg gaggcctcag tctgttctct  16879 

taaggccttc aactgattaa atgaggcctg cccaagttat agagagtaac ctgccttact  16939 

ccgtcttctg atttaaatgt tagtcacatc taaaaaatat tttcgcagca gcatttccac  16999 

tggcttttga ccaaacatca ggccacaaag ttgatcccca aaattaacca tcactctgtg  17059 

cctgtaaggg aggggctggg aaaggggagc aggtctcccc aaggggtgac cttggctttg  17119 

ttcctcccag gc  ctg gag ttt att cgg aaa agc cag ctg gtc cag cct     17167 
           Gly Leu Glu Phe Ile Arg Lys Ser Gln Leu Val Gln Pro 
                       270                 275 

gtg ggg cca ctg gtg gtg ctg ctg ccc ctg gcg ggt ggg tac agc cgc    17215 
Val Gly Pro Leu Val Val Leu Leu Pro Leu Ala Gly Gly Tyr Ser Arg 
280                 285                 290                 295 

gtc ctc aac gcc gcc tgc cag cgc ctg gcg agg gct ggg gtc gtg ctg    17263 
Val Leu Asn Ala Ala Cys Gln Arg Leu Ala Arg Ala Gly Val Val Leu 
                300                 305                 310 

gtc acc gct gcc ggc aac ttc cgg gac gat gcc tgc ctc tac tcc cca    17311 
Val Thr Ala Ala Gly Asn Phe Arg Asp Asp Ala Cys Leu Tyr Ser Pro 
            315                 320                 325 

gcc tca gct ccc gag gtaggtgctg gggctgctgc cccaaggcgc gggtaggggg    17366 
Ala Ser Ala Pro Glu 
        330 

cggagggcgg agggagggcg ggcgggcagg cgggcttctt gtggcacgtg ggcttcttgt  17426 

ggcacgttcc tggaggccga acccttctgg ctttggaagg agtcgtcaga gacccccgcc  17486 

atgcgggagg ctggggagga aggggctcga aacctccatc atcgcagagt ctgaatagca  17546 

gtggccccgc catgcgccca cgtagcggcg cctacgtagc cacgccccca cgccccgtcc  17606 

tggccactct ccctcctgaa ggtcttctgg tacccgcccc ctccccatct ccatccccag  17666 

gccctgcgtc ctctgcccaa tactctttgg gcctccctgt tgtccagctc tctccgcggc  17726 

tccatgactg acaacttgag caaggctaat gtgaatggga gcggttgagg gctcagacct  17786 

ctcacccgag gaacatccac agagtgtgcc gcatgcccgg tgcagtgtgg ctgcggggac  17846 

acagacacgg agcctcggcc ctgaggagct ggggggcagt gaccgtccct cctctgaccc  17906 

accactcctc cagtgtcagg acactgcggg tatctagggg aaggaatctt gttccacttc  17966 

aagtctggaa cttcaagtct gtgtgtgtgc gtgcgcgcgc gcgcgttggg ggtgggggtt  18026 

gcagagcaga tgcgtacctg acagcggtaa cctaggtccc cccggcctat caaggcttcc  18086 

ctggcggccg aatttaaagg catcaagcaa acaaagccca acacatctct gccttgtcct  18146 

ctcagtttcc ccccgtggca cttagaacca cttgatacac cgaatagttt ccggtctatc  18206 

tcccccacta ggatgtaaac tccacagggg cattgggaat gctgcctggc tatggtaggg  18266 

acagagggga gcaccagggc ggggcagggg tgccagagtt ctgcctgggc agtcagattt  18326 

tccttaggag gggacatttg agtgggaccc aaacaggtgt atagcagttg tccagcccag  18386 

ctggcaaggc ctgagtctgc ctctgcaacc cctctcttgg gctcctttct ctgccaccca  18446 

cctcctcacc tttccag gtc atc aca gtt ggg gcc acc aat gcc cag gac     18496 
                   Val Ile Thr Val Gly Ala Thr Asn Ala Gln Asp 
                           335                 340 

cag ccg gtg acc ctg ggg act ttg ggg acc aac ttt ggc cgc tgt gtg    18544 
Gln Pro Val Thr Leu Gly Thr Leu Gly Thr Asn Phe Gly Arg Cys Val 
    345                 350                 355 

gac ctc ttt gcc cca ggg gag gac atc att ggt gcc tcc agc gac tgc    18592 
Asp Leu Phe Ala Pro Gly Glu Asp Ile Ile Gly Ala Ser Ser Asp Cys 
360                 365                 370                 375 

agc acc tgc ttt gtg tca cag agt ggg aca tca cag gct gct gcc cac    18640 
Ser Thr Cys Phe Val Ser Gln Ser Gly Thr Ser Gln Ala Ala Ala His 
                380                 385                 390 

gtg gct g gtaagtcacc accccactgc ctcggccacc gtgatgctaa cagccccttt   18697 
Val Ala 

ggcagtcagg gtctgtgccg ggacctccag tgccaggctc tgtgcagggg gaccagagat  18757 

gaagtaggcc tgatggtgcc ttcaaggaca ctcagtctga tgagggaggc gagtgcacag  18817 

agggaacacg aggtcagggc tgtattagag ggagcccaga ggaggcacct gcccagcccg  18877 

agggtcagag aaggcatctt ggaggaggga catttgatcg ggagcttgat ggatgaatag  18937 

gagtttacct ggccgataag acagcaacta ccaaggctta gaggtgtgag aggaggctgt  18997 

cttacctcac tgagtaagga ctgcaggcgg cttaccttcg agaagagagc ttagtgtctg  19057 

tgtgcacgtg tgtttgtgtg tatgtgtgtg cgtgtgtgca ctggcaggag tcccctgctg  19117 

gggcaggagg gccgggccat caccatcttt caccattcac ccctgcacca ggc att    19173 
                                                       Gly Ile 
                                                           395 

gca gcc atg atg ctg tct gcc gag ccg gag ctc acc ctg gcc gag ttg    19221 
Ala Ala Met Met Leu Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu 
                400                 405                 410 

agg cag aga ctg atc cac ttc tct gcc aaa gat gtc atc aat gag gcc    19269 
Arg Gln Arg Leu Ile His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala 
            415                 420                 425 

tgg ttc cct gag gac cag cgg gta ctg acc ccc aac ctg gtg gcc gcc    19317 
Trp Phe Pro Glu Asp Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala 
        430                 435                 440 

ctg ccc ccc agc acc cat ggg gca g gtaagcagga tggcagggtg            19362 
Leu Pro Pro Ser Thr His Gly Ala 
    445                 450 

ggcaagtcca ggctggggct tgggaggtct gtgtgacctt gacagtctct cccttctccc  19422 

ttgtctgtgt aaggaggatg atgccacctt aaataggatt aaatgagaat ggggctctga  19482 

aagggctgtg caatattttc ataacgtgtt tttatagaga cagttgagta tgttctttaa  19542 

gccctcctct ctcctaccat gaactaaaga tttttgtgga ggtcccctca ctcccagcac  19602 

cccctcctca tcccaggccc tttttgca ggt tgg cag ctg ttt tgc agg act     19654 
                               Gly Trp Gln Leu Phe Cys Arg Thr 
                                           455 

gtg tgg tca gca cac tcg ggg cct aca cgg atg gcc aca gcc atc gcc    19702 
Val Trp Ser Ala His Ser Gly Pro Thr Arg Met Ala Thr Ala Ile Ala 
460                 465                 470                 475 

cgc tgc gcc cca gat gag gag ctg ctg agc tgc tcc agt ttc tcc agg    19750 
Arg Cys Ala Pro Asp Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg 
                480                 485                 490 

agt ggg aag cgg cgg ggc gag cgc atg gag gtgactgtac ccctccttcg      19800 
Ser Gly Lys Arg Arg Gly Glu Arg Met Glu 
            495                 500 

tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgcgtgtcag tgctgggccc tcagggaccc  19860 

ccagcaagcc cctccatcct ccagactcca gctcttctgt aagcttacag ggctggccag  19920 

accaggagtg gggcactcct cacttcacgc ggctgggggc tgctggagag agccacagcg  19980 

ggaagggttt cctagaggct gcaggacagt gctggatgga ttttcaatgc tcacctgggt  20040 

gtgagcatgc ggcagggccg cgtgagggtc agcgatctgc tactctggac tcagccatct  20100 

ctaggcccct ctcactcagg tgctccatgg ttctgggagc tgagaaatct caaaccagca  20160 

aaaaagtgga attgatgttg atgctacagg atagtgcaca gatgccatct ggttgcagca  20220 

ttttggtgga agggcagtgc ccagctagga gagtgaggag gggcaggcat ttctggcttg  20280 

aggagatagg gtcttaatgc tcgtgtgaga ggcagagtgg gtggagtgga gctggctgga  20340 

tccttgcttt ggcctcctgg atttctctct atctccattt tgaaaccact ctgtgtttgg  20400 

aagaactttt gagtattcag agctgcccac tggcagaaca gtcttccttg ggcaggagtg  20460 

agctccttgt ccccagaagg ctgggtctgg ctggcccctg gcagggacac tgatgagggt  20520 

gcttgagttg atcctgtcta gtccctttct gtgttttcaa agcccattct aaagcagatt  20580 

cccatttccg tctttgactc taag gcc caa ggg ggc aag ctg gtc tgc cgg     20631 
                           Ala Gln Gly Gly Lys Leu Val Cys Arg 
                                       505                 510 

gcc cac aac gct ttt ggg ggt gag ggt gtc tac gcc att gcc agg tgc    20679 
Ala His Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys 
                515                 520                 525 

tgc ctg cta ccc cag gcc aac tgc agc gtc cac aca gct cca cca gct    20727 
Cys Leu Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala 
            530                 535                 540 

gag gcc agc atg ggg acc cgt gtc cac tgc cac caa cag ggc cac gtc    20775 
Glu Ala Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val 
        545                 550                 555 

ctc aca g gtaggaggct gggcttgccc tggggtgagg aggggtctct ttctccttat   20832 
Leu Thr 
    560 

gcacccactg cccacgaggc ttggtcctca caagtgtgat ccatgagact caagcctgac  20892 

ttgcagttcc atactctggt tctgccactt ccatgccctt tgagcctggg caggtgacct  20952 

tacttctcct catctcagct tcctcctcca taagagggaa aaaggtatta cctgcctcat  21012 

tgtgttgcaa ggagatgggc agcatctagg gcactggcct ggagtatcgc aggtgctttg  21072 

cctaaggtgg tgcagtccag gagaggcagc tccagagaga ggcccccggc tggggctgaa  21132 

aggagggcag acctcggttt gaatttcacc ctgccgctcg atagctgtgt gacttgggca  21192 

aattacttaa catctctgta tgaggaaatg atgagtgcta agcacttagc ttagtgccgg  21252 

gacaatataa attctagcta tcgttactat tgttttcatc acccgttgct ttaaaatcca  21312 

gtctctggta taggcaacta ttgacgggct accctgtgtc gaaaacatgc ccaggcaggt  21372 

agcaggaagt cacagatggg gacctcttgg ggcatcaagg gatggtgccc tgaggctgag  21432 

ctgttctggt tgggtggagc atgagaggtc tgggaagaca gtgggactcc agcctggaat  21492 

aagaggctca gagttgattc tcgtctgagc acgtccaggg gaaccactga gggtttggga  21552 

acaggagagt gagggtgaga acctggttct gggcacagca ggctggcatg taggatggat  21612 

gttcaggaaa gatgagcata gtcaggtggc tggtgccctt gtccagggga gaggctccgt  21672 

caggttcagg ggtcctggct tggagggaag tccgccatgc tctaatcacg ctcccctttg  21732 

gaagtgctcg gccgatgagc tcacaggcac atgtcagttt gaagtcatgg aatctgactc  21792 

catgaagcgc acctcaaaga gcaccatttt gcagctaagg gaactgcagg ctggacatgc  21852 

tgagtggctg ccccgagccc ttgcagctag gacatagaga atgctagtaa ccacaaccct  21912 

accatgttca gagcacatgc caggctccat gctggggctt cgcacgtgtc atcttcacag  21972 

tgtccctgtg agtaggtgtg gtttctcttt ccatcttaca aatgagtaaa cagagcctca  22032 

gtgtagctaa gtaaccacta ttttaggttt cttagccaat gggtgtgtct gactcctaag  22092 

cccatggagg gcattctgag gtggttcaga cagaccccag cttacccttg aacttctgcc  22152 

tgctggctgc atagggaggg gctgggggga gtttgagcat ctcaggccat agagcccctg  22212 

cctcactgtc tccatctctg ggtggaaaga tggtgttttc cctgagaaac taaggctcag  22272 

agaggttgaa tggctctccc aaggtcacac agctggtcag ctgcagagtt gagaacacag  22332 

gagtcctggt gctcaggcca gcatctcttt ttttctttga gttgtttcta ggtttcctag  22392 

ctcttgcctc agaccttaaa gagagagggt ctgatgggga tgggcactgg agacggagca  22452 

tcccagcatt tcacatctga gctggctttc ctctgcccca ggc tgc agc tcc cac    22507 
                                            Gly Cys Ser Ser His 
                                                            565 

tgg gag gtg gag gac ctt ggc acc cac aag ccg cct gtg ctg agg cca    22555 
Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro Pro Val Leu Arg Pro 
                570                 575                 580 

cga ggt cag ccc aac cag tgc gtg ggc cac agg gag gcc agc atc cac    22603 
Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg Glu Ala Ser Ile His 
            585                 590                 595 

gct tcc tgc tgc cat gcc cca ggt ctg gaa tgc aaa gtc aag gag cat    22651 
Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys Lys Val Lys Glu His 
        600                 605                 610 

gga atc ccg gcc cct cag gag cag gtgaagaggc ccgtgaggcc gggtgggtgg   22705 
Gly Ile Pro Ala Pro Gln Glu Gln 
    615                 620 

ggtgctgcgt gtctctcctg cacagctttt ctgtgtcagt ttgtgccacc accataccgc  22765 

catacatcag ggtggcggtt tgccaggtag atgctgtggg cagcttccgc cattgtgtgg  22825 

acagcatgta tatgtgtctc tgtgtggctg ggtctgtttt tgcttttgtc cagatcagta  22885 

aggtttgcta cctgggtacc ccactccact tggagtagag tgtgcataaa tatggcataa  22945 

agaaatgcaa tatgcatgca tttattgatt gatctatttt tttctgagat ggggtcttgc  23005 

tgtgttgccc aggctggtct caaattcctg ggctcaagca atcctctggt ctcagcctcc  23065 

ccaagtgttg ggattatagg catgagccgc tgcacctggc ctctctgatc tatttaacaa  23125 

acctgctggg agggtctcag ggtcaggagc agcactgggc tctgaggaca cagagctcac  23185 

tcagccgtga cccagagggg gtgcctgagc tgcatgctga aggttgttag catgaccagc  23245 

aaggcaagaa aaggccctgc cgagattagc aaggcatgtg ccaagccctg gaatgtgaca  23305 

gccgggcctt ctagaaacct gagtgtataa ctctccttaa aagccagtag gagctcctta  23365 

aaaggcagcc ctaaggagtc cactcttaaa tgaactcaga gtcagtttta aaatgcaagt  23425 

ctgtgttgat tctggtctgg atggtgcatt cctcgagagc aaaagacagt cttggtcttg  23485 

gatccacttg ccctgggtac actgagggct gctaggttcc aggtgctctt cctggcactg  23545 

gggagggata caggcccaag agacatgctg ttctccctcc tggagcatct attttagtgg  23605 

aggaagacag aaaacaaacc attaatatag agtactgaaa agatgcgatg gagaaaacta  23665 

tagcaaggaa gggaatgggg tgggagagag gtcaggagag gtctcgctga caaggtggac  23725 

gaaacaggcc atgaggcaga gaacatgttc caggcaaagc aaaggccccc aggtggggat  23785 

gtgcagggag taccaggaaa ccagagaggt gggaatagtt atgagatggg gggtgcctca  23845 

gaggggacag ggccaagtca ggtgagacct gagggccaca gtcagcagtg agctggggcc  23905 

atgcaggggt ctggcctcag aggagtgtgg tctggcctgg atctgaacct ctcactgtgg  23965 

cctagctgct gagctgagaa gagatgacaa ggaccttggg cagaagcagg gagactggag  24025 

ggaggcggtg gagggtccag gcgttggggc ggggctcagg ctggagtctg aagggagcct  24085 

gcaggcctgg tgggtggatg tgggtgggag agggggagga tggcaccaag gctcgggccc  24145 

ctggacagat ggagttgcca ttaagtggga tggggcaggc tatggggcca tcagtttcag  24205 

agggatgagt ttggcactgg catggtaggc atctgtctat ctccacggcc ctcaaaccag  24265 

gcatgaagca ggagctcacg tgtttggtca gccatggtgc agaaccgcct gggtgggagg  24325 

tgcggggtgg gagatacacg gttgtgtccc aaatgggctc tgagccagcg agggccgtct  24385 

gcactttggc ctcacagaag gatgtcggag ggagaaatga agtgtgggtg ggggtcccgg  24445 

gccacgctag acatgtgctt tcttttcctc gggctctggc ag gtg acc gtg gcc     24499 
                                               Val Thr Val Ala 
                                                           625 

tgc gag gag ggc tgg acc ctg act ggc tgc agt gcc ctc cct ggg acc    24547 
Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly Thr 
                630                 635                 640 

tcc cac gtc ctg ggg gcc tac gcc gta gac aac acg tgt gta gtc agg    24595 
Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val Arg 
            645                 650                 655 

agc cgg gac gtc agc act aca ggc agc acc agc gaa gag gcc gtg aca    24643 
Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Glu Ala Val Thr 
        660                 665                 670 

gcc gtt gcc atc tgc tgc cgg agc cgg cac ctg gcg cag gcc tcc cag    24691 
Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser Gln 
    675                 680                 685 

gag ctc cag tga cagccccatc ccaggatggg tgtctgggga gggtcaaggg        24743 
Glu Leu Gln 
690 

ctggggctga gctttaaaat ggttccgact tgtccctctc tcagccctcc atggcctggc  24803 

acgaggggat ggggatgctt ccgcctttcc ggggctgctg gcctggccct tgagtggggc  24863 

agcctccttg cctggaactc actcactctg ggtgcctcct ccccaggtgg aggtgccagg  24923 

aagctccctc cctcactgtg gggcatttca ccattcaaac aggtcgagct gtgctcgggt  24983 

gctgccagct gctcccaatg tgccgatgtc cgtgggcaga atgactttta ttgagctctt  25043 

gttccgtgcc aggcattcaa tcctcaggtc tccaccaagg aggcaggatt cttcccatgg  25103 

ataggggagg gggcggtagg ggctgcaggg acaaacatcg ttggggggtg agtgtgaaag  25163 

gtgctgatgg ccctcatctc cagctaactg tggagaagcc cctgggggct ccctgattaa  25223 

tggaggctta gctttctgga tggcatctag ccagaggctg gagacaggtg tgcccctggt  25283 

ggtcacaggc tgtgccttgg tttcctgagc cacctttact ctgctctatg ccaggctgtg  25343 

ctagcaacac ccaaaggtgg cctgcgggga gccatcacct aggactgact cggcagtgtg  25403 

cagtggtgca tgcactgtct cagccaaccc gctccactac ccggcagggt acacattcgc  25463 

acccctactt cacagaggaa gaaacctgga accagagggg gcgtgcctgc caagctcaca  25523 

cagcaggaac tgagccagaa acgcagattg ggctggctct gaagccaagc ctcttcttac  25583 

ttcacccggc tgggctcctc atttttacgg gtaacagtga ggctgggaag gggaacacag  25643 

accaggaagc tcggtgagtg atggcagaac gatgcctgca ggcatggaac tttttccgtt  25703 

atcacccagg cctgattcac tggcctggcg gagatgcttc taaggcatgg tcgggggaga  25763 

gggccaacaa ctgtccctcc ttgagcacca gccccaccca agcaagcaga catttatctt  25823 

ttgggtctgt cctctctgtt gcctttttac agccaacttt tctagacctg ttttgctttt  25883 

gtaacttgaa gatatttatt ctgggttttg tagcattttt attaatatgg tgacttttta  25943 

aaataaaaac aaacaaacgt tgtcctaact cttgcataga cttgactgcc tagggtgatg  26003 

ccttgcttat actaggaact gggtaagttt gttgaatagt tgagtaagcc aagtatttga  26063 

tgagtacttg tatcttgagt acaagtattg ggcaagtact ggtgatgtga acttactcct  26123 

tgtgcctatc ctaggaatga aatgaatgtc ttcctgcagc tcccctgacc accctgacag  26183 

tcaaagtgcc tcctccttgg tgacaggtgc cctacag                           26220

Technology Category: 1