Nucleotide sequence which encodes a flavin monooxygenase, the corresponding protein and their uses in the spheres of diagnosis and therapy

The present application is a continuation of PCT application Ser. No. PCT/FR/97/02226, filed Dec. 5, 1997 (the disclosure of which is incorporated herein in its entirety) which claims priority from French Patent Application Serial Number 96/15032, filed Dec. 6, 1996, the disclosure of which is incorporated herein by reference in its entirety.

The present invention relates, in particular, to human flavin monooxygenase 2 (hFMO2), as well as to another human enzyme of the FMO family, i.e. hFMOx, and to their nucleotide and polypeptide sequences. The present invention also relates to cloning and/or expression vectors which contain said nucleotide sequences and to cells which are transformed with these vectors, as well as to methods for preparing said polypeptides. The invention also encompasses methods for selecting compounds and for diagnosing predisposition to pathologies and/or deficiencies which are linked to the FMOs as well as to pharmaceutical compositions which comprise said compounds, which are intended for treating and/or preventing these pathologies.

The flavin monooxygenases (FMOs) (Lawton et al., 1994) form a family of microsomal enzymes which catalyze the NADPH-dependent oxidation of a large number of exogenous organic compounds (xenobiotics) which possess a nucleophilic heteroatom such as, in particular, the nitrogen, the sulfur, the phosphorus or the selenium atom (Ziegler D. M., 1988; Ziegler D. M., 1993), whether the xenobiotics are drugs, pesticides or other potentially toxic substances. Cysteamine is currently the only known endogenous substrate of the FMOs.

The FMOs represent a multigenic family. Expression of different forms of FMO depends both on the tissue and the species under consideration.

FMOs have been located in various types of tissue, in particular the liver, the lungs and the kidneys.

To date, five isoforms of FMO have been characterized in the reference species, which is the rabbit. Their homology is 50-60%. Four of these isoforms, i.e. FMO1, FMO3, FMO4 and FMO5, have been identified in humans (GeneBank sequences M64082, M83772, Z11737 and L37080, respectively). Among the mammalian species, the homology between orthologous FMOs is greater than 80%. It is reasonable to postulate that an FMO2, if not to say other isoforms, exist(s) in humans.

The FMOs are associated with the endoplasmic reticulum and are involved in detoxifying xenobiotic compounds, with monooxygenation enabling the xenobiotic to be transformed into a more polar substance, with this transformation being the preliminary step prior to its excretion. The FMOs may also be involved in the metabolic activation of various toxic and/or carcinogenic compounds which are present in the environment.

The mechanism of the FMO reaction has been described in detail (Poulsen, L. L. et al., 1995). In contrast to all the other known oxidases or monooxygenases, the FMOs possess the unique property of forming a stable, NADP(H)- and oxygen-dependent enzyme intermediate, i.e. 4α-hydroperoxyflavin, in the absence of oxidizable substrate. Because the catalytic energy is already present in the FMO enzyme before contact with its potential substrate, the appropriateness of the substrate does not have to be as precise as in the case of other types of enzyme. This specific characteristic of FMO is responsible for the large variety of substrates which are accepted by the FMOs (including, for example, tertiary and secondary alkylamines and arylamines, many hydrazines, thiocarbamides, thioamides, sulfides, disulfides and thiols).

Many molecules which are active compounds of drugs are recognized as being substrates of the FMOs, either for an N oxidation or for an S oxidation (Gasser, 1996), with these molecules including, in particular, antidepressants, neuroleptics, anti-ulcer drugs, vasodilators and antihypertensives.

Although some FMO substrates are oxidized into less active derivatives, a large number of nucleophilic compounds can be metabolized into intermediates which may be more reactive and/or potentially toxic; rather than being excreted, such products may induce toxic responses by means of covalent binding to cell macromolecules, or by means of other mechanisms. For example, mercaptopyrimidines and thiocarbamides may be mainly activated by FMO activity (Hines et al., 1994). More precisely, it has been demonstrated that the nephrotoxicity which is associated with the glutathione conjugate of acrolein is linked to its metabolism mediated by renal FMO; the FMO forms an S-oxide which is then released, by an elimination reaction which is catalyzed in basic medium, in the form of cytotoxic acrolein (Park, S. B. et al., 1992). Thus, the FMOs can play an important role both in the first steps of chemical toxicity and in the detoxification of xenobiotic compounds.

As described above, a large number of drugs which are currently at the clinical trial stage, or else widely prescribed, contain nucleophilic functions of the nitrogen, sulfur, phosphorus or other type. However, the role of FMO in the oxidative metabolism of drugs and endogenous chemical compounds in humans is not well understood.

Cashman et al. (1996) have recently studied the contributions of the FMO enzymes in the physiological metabolism of cimetidine and S-nicotine in vivo. The greater part of their results confirms the fact that the FMO3 activity of the adult liver is responsible for the oxygenation of cimetidine and S-nicotine, with this oxygenation being stereospecific. The authors furthermore demonstrate that the stereochemistry of the main metabolites of cimetidine and S-nicotine in small experimental animals is different from that observed in humans and suggest that different FMO isoforms may predominate depending on the species, with this possibly having important consequences with regard to the choice of experimental animals for programmes for elaborating and developing drugs for humans.

FMO1 is known to be expressed in humans in the kidneys but not in the liver. FMO2 is expressed in the main in the lungs in all the mammalian species tested. In humans, FMO3 was isolated from the liver, where it predominates in adults. FMO3 is the main isoform involved in the sulfoxidation of methionine and in the stereospecific oxygenation of cimetidine and S-nicotine. FMC3 exhibits a greater specificity for its substrate than that exhibited by the FMO1 enzymes which are found in the livers of most animal species studied. FMO4 is a minor isoform whose function and substrate specificity are not well known. It is present in the human liver and is also expressed in the brain, where it could be involved in the oxidation of antidepressant drugs such as imipramine. FMO5 is expressed in the human liver to a lower extent than is FMC3. Its apparent lack of efficacy as an enzyme involved in the metabolism of drugs suggests that it could be involved in a physiological function.

The differing expression profiles of the FMO isoforms, depending on tissues and/or species, therefore probably constitute a significant factor contributing to the differences in FMO activity which are observed between tissues and/or between species. Thus, the variety of FMO forms could have a significant impact on the differences in the responses of tissues and/or species to exposure to a xenobiotic compound. This is because the differences which are observed between tissues and/or species in the response to xenobiotic compounds, and in the toxicity of these compounds, are linked, to a substantial extent, to variations in the activity and specificity involved in the metabolism of these substrates by the FMOs. Genetic factors and tissue specificity in the expression of the FMOs are important factors in these variations.

With regard to genetic factors, it has been reported, for example, that trimethylaminuria, which is a condition which is present in 1% of white British subjects and which is expressed in a strong odor of rotting fish in the expired air, the sweat or the urine, is linked to a deficiency of genetic origin in the functioning of an hepatic FMO.

For the reasons which have previously been mentioned, there therefore currently exists a considerable need to identify new isoforms of FMO, as well as the genetic polymorphisms which may be associated with them, which exhibit specificities with regard to their substrates and/or their tissue expression profile, which could be involved in the metabolism of xenobiotics, such as the metabolism of drugs or of exogenous substances which are present in the environment, such as, for example, pesticides, or else which could be involved in a physiological function. This is precisely the object of the present invention.

FIG. 1 : Analysis of the segregation of the G.1263mac.A polymorphism in the family studied.

The genomic DNA of individuals 3, 4 and 7 to 14 was amplified by PCR and the sequences of the resulting fragments were analyzed in order to detect heterozygosity sites which segregated with the disease.

The filled-in symbols indicate the individuals suffering from juvenile POAG. The barred symbols indicate individuals who were not genotyped. Individuals 11 and 12 are twins.

G/G=homozygotes for the base in the position which is homologous to position 1263 of the macaque FMO2 mRNA.

G/A=heterozygotes for the base which is in the position which is homologous to position 1263 of the macaque FMO2 mRNA.

Table 1 depicts primer sequences which can be use for amplifying the sequences which are of interest in relation to the G.1263mac.A. mutation.

Table 2 lists examples of primers which can be used for detecting the G.1263mac.A mutation by Single nucleotide primer extension.

Table 3: Example of a restriction enzyme which can be used for detecting the G.1263mac.A mutation by Restriction Fragment Length Polymorphism (RFLP).

Table 4: Examples of probes which can be used for detecting the G.1263mac.A mutation by allele specific oligonucleotide (ASO).

Table 7A: Description of the exon/intron structure of the gene which encodes hFMO2, which is the human homologue of macaque FMO2. The positions where the exons begin and end are shown on the nucleotide sequences SEQ ID No. 1 and No. 2.

Table 7B: Description of the exon/intron structure of the gene encoding hFMOx. The positions where the exons begin and end are shown on the nucleotide sequences SEQ ID No. 4 and No. 5.

Table 8: Homology between the macaque FMO2 gene and its human homologue. The 5′ untranslated region diverges slightly from the macaque sequence.

Table 9: Summary of the positions at which the human hFMO2 mRNA sequence varies as compared with the homologous macaque sequence; influence of the variations on the protein sequence.

Several genes of the human FMO family have been located on the 1q23-25 region of chromosome 1 by means of in situ hybridization of the metaphase chromosome.

Once such a candidate region has been defined, it is necessary to have access to the fragment of the genome which covers the distance over which the sought-after gene(s) is/are located. This step proceeds through the drawing up of a physical map, namely the covering of the region with a set of cloned and ordered fragments. At present, thanks to the data of the CEPH/Généthon integrated map of the human genome, approximately 80% of the genome is covered by YAC clones which are subcloned into BACs whose location on the chromosomes is determined by means of polymorphic and genetically ordered markers (Chumakov et al., 1995). This physicogenetic map makes it possible to save a considerable amount of time, in particular by the use of exhaustive sequencing of the regions of interest.

Thus, according to the present invention, it was established, after locating the BAC 123H04M on the previously mentioned genetic locus 1q24-25, that the insert which it carries contains the 3′ part of hFMO3 and the 5′ part of hFMO1 as well as the complete sequence of hFMO2 and that of another new gene which is a member of the FMO family, i.e. hFMOx.

Furthermore, as a result of using 5′ label libraries, it is possible to verify the expression of the candidate genes which have been identified as above: the identification of a label which hybridizes to one of the candidate sequences indicates, since this sequence is derived from a cDNA library, the presence of mRNA and therefore of expression of the sequences in question in the tissues under consideration.

For this reason, the present invention relates, in particular, to an isolated polynucleotide whose sequence, i.e. SEQ ID No. 1, which encodes a polypeptide having the sequence SEQ ID No. 3.

The present invention also relates to an isolated polynucleotide whose sequence, i.e. SEQ ID No. 4, which encodes a polypeptide having the sequence SEQ ID No. 6.

These two nucleotide sequences are those of two genes which encode novel enzymes of the human flavin monooxygenase (FMO) family, i.e. hFMO2 and hFMOx, respectively. This was established by comparing the identified sequences with the previously known FMO sequences: the conclusion was made possible by very strong structural homologies between the two sequences studied and those of the FMOs, very strong homologies between the first sequence and the known FMO2s, in particular the macaque FMO2 (macaque FMO2: GeneBank sequence U59453), as well as insufficient homology of the second sequence with any of the FMOs which have already been itemized in humans.

The exon structure of the already known genes of the FMO family is entirely conserved in the hFMO2 nucleotide sequence according to the invention. The sequences of each of the 9 exons of the polynucleotide according to the invention (Table 7) exhibit degrees of DNA homology varying from 95% to 98% with the corresponding sequence of the messenger RNA of the macaque FMO2 (Talbe 8). The divergences between the two nucleotide sequences, as well as their significance for the peptide sequence, are shown in Table 9. The polynucleotide sequence SEQ ID No. 1 according to the invention encodes a polypeptide of 535 amino acids having the sequence SEQ ID No. 3; the sequence SEQ ID No. 2 of the predicted messenger RNA, as well as the polypeptide sequence of the human protein, are 97% homologous with those of the macaque FMO2, thereby making it possible to identify the polypeptide according to the invention as being human FMO2. The polypeptide having the sequence SEQ ID No. 3, also exhibits a high degree of homology with other mammalian flavin monooxygenases 2; its degrees of homology with other proteins of the flavin monooxygenase family are lower.

As previously mentioned, the lack of sufficient homology between the sequences corresponding to hFMOx—genomic (SEQ ID No. 4), messenger RNA (SEQ ID No. 5) and peptide (SEQ ID No. 6) sequences—and the sequences of the known FMOs enabled the conclusion to be drawn that hFMOx is a novel FMO isoform.

The present invention therefore relates to the DNA or RNA sequences, with the DNA being able to be genomic DNA, complementary DNA or synthetic DNA, of the FMOs, in particular of hFMO2 and hFMOx, as well as to the corresponding proteins.

The present invention furthermore relates to cloning and/or expression vectors which contain said nucleotide sequences, to cells which are transformed with these vectors or to animals which contain said cells, as well as to methods for preparing said polypeptides in the form of recombinant polypeptides.

The invention also encompasses methods for selecting a compound which is able to modulate FMO activity.

The invention also relates to methods for diagnosing predisposition to FMO-linked disorders as well as to pharmaceutical compositions which are intended for treating and/or preventing these disorders.

A first example of such disorders could be primary open-angle glaucoma (POAG). Thus, on the one hand, Sunden et al., (1996), as well as the inventors (Belmouden et al., 1996), have identified the chromosomal region GLC1A, which carries, among other gene sequences, those known sequences of the FMO family, in 1q23-25, as being linked to the appearance of juvenile POAG (J-POAG). On the other hand, a possible role for monooxygenases in the etiology of glaucoma has previously been suggested (Schwartzman et al., 1987). Thus, it has been suggested that, by inhibiting the Na+, K+, ATPase activity in the cornea, oxidation reaction metabolites might contribute to regulating the transparency of the cornea and ocular humoral secretion; it should be noted that opacity of the cornea and ocular hypertension are the two main criteria for diagnosing glaucoma.

Thus, the inventors have identified a site of heterozygosity, exhibiting genotypic segregation in a family studied for the presence within it of a large number of members suffering from J-POAG, in exon 8 of the hFMO2 polypeptide according to the invention.

By looking for polymorphisms which are present in appropriately selected populations and which are located in sequences which correspond to those carried by the BAC 123H04M insert, or more generally by the FMO sequences, it will be possible to identify, in particular, the mutations which are associated with pathologies or disorders which are linked to an alteration in the FMOs.

The various FMO isoforms appear to differ from each other less by the tissue specificity of their expression than by the substrates whose transformation they catalyze. As previously pointed out, FMOs have been shown to be expressed in the liver, the lungs, the kidneys and the brain.

The pathogenic effect of a functional deficit in an FMO could result in a decreased capacity of the tissues, in which it is expressed, to resist oxidative stress.

More generally, as a result of their role in oxidative metabolism and their detoxification function, the FMOs could be involved in any degenerative or toxic pathology which has been demonstrated or is still to be proved, in particular those pathologies in which programmed cell death has been shown to take place, and the degenerative diseases of the central nervous system.

In a general manner, the pathologies linked to FMO function are grouped under the name “FMO-linked disorders”.

FMO-linked disorders which may be mentioned by way of example, but without any limitation to these disorders, are:

oxidation of drugs, which are FMO substrates, to form less active derivatives, implying a loss of efficacy of said drug;

failure to metabolize drugs which are active in metabolite form; loss of efficacy of said drug;

failure to metabolize toxic and/or carcinogenic xenobiotics, including exogenous substances which are naturally present in the diet, such as plant alkaloids, or toxic substances which are present in the environment, such as pesticides or herbicides;

metabolism of drugs to form intermediates which may be more reactive, implying overdosing with the possibility of side-effects;

metabolism of xenobiotics, including drugs or other exogenous substances, to form intermediates which may potentially be toxic; and/or

alteration of the physiological function in which the FMO is involved; in particular alteration of FMO function could be involved in the symptomatology of glaucoma.

“FMO” will be understood as referring to any human FMOs which are known, i.e. FMO1, FMO3, FMO4 and FMO5, or which are newly described in the present patent application, namely FMO2 or FMOx.

While some of these disorders may have a multigenic origin, it applies to all of them that alterations to one or more FMOs contribute to the appearance of the disorder or to its aggravation.

The Nucleotide Sequences

The present invention first of all relates to an isolated nucleotide sequence which is distinguished in that it is selected from:

a) the sequences which encode the human FMO2 or FMOx proteins and their protein variants,

b) the sequences which encode a fragment of these proteins which possesses at least 10 bases,

c) the human FMO2 or FMOX genomic sequences and their alleles,

d) the sequences which exhibit at least 80%, preferably at least 90%, homology with the sequences (a) and (c),

e) the fragments of the sequences (c) or (d) which possess at least 10 bases,

f) the sequences which hybridize with a sequence from (a) to (e).

It should be understood that the present invention does not relate to the genomic nucleotide sequences in their natural chromosomal environment, that is to say in their natural state; the present invention relates to sequences which have been isolated, that is which have been picked out directly or indirectly, for example by making a copy (cDNA), with their environment having been at least partially modified.

Thus the sequences to which the invention relates can just as well be cDNA as genomic DNA which is partially modified or carried by sequences which are at least partially different from the sequences which carry them naturally.

These sequences can also be described as being “unnatural”.

A “nucleic acid sequence” is understood as being a natural, isolated, or synthetic, DNA and/or RNA fragment which designates a precise sequence of modified or unmodified nucleotides, which sequence makes it possible to define a fragment, a segment or a region of a nucleic acid.

“Alleles” are understood as referring to the mutated natural sequences which correspond to polymorphisms which may exist in the human being, in particular those which may lead to the development of FMO-linked disorders.

“Protein variant” is understood as referring to the entirety of the mutated proteins which may exist in the human being and which correspond, in particular, to truncations, substitutions, deletions and/or additions of amino acid residues, as well as the artificial variants which will nevertheless also be termed “protein variants”. In the present case, the variants are linked in part to the occurrence of FMO-linked disorders.

According to the invention, the fragments of nucleic acid sequences may, in particular, encode domains of the protein or else be used as probes or as primers in detection, identification or amplification methods. These fragments are at least 10 bases in size, and preference will be given to fragments which contain 20 bases, preferably 30 bases.

According to the invention, the homology is solely of the statistical type; it signifies that the sequences possess at least 80%, preferably 90%, of their nucleotides in common.

As far as the (f) sequences are concerned, the hybridization conditions should ensure, according to the invention, at least 95% homology.

More specifically, the present invention relates to a nucleotide sequence which is selected from:

a) the sequences which encode a polypeptide which comprises the amino acids according to the sequence SEQ ID No. 3 or according to the sequence SEQ ID No. 6,

b) the nucleic acid sequences of SEQ ID No. 1 or No. 2, or the nucleic acid sequences of SEQ ID No. 4 or No. 5, or the nucleic acid sequences which encode the corresponding polypeptides,

c) a fragment of a sequence according to (a) or (b) which contains at least 10 bases, and

d) a sequence which contains at least one point mutation as compared with the sequences (a), (b) or (c),

e) a sequence which is complementary to the sequences (a), (b), (c) or (d).

The structure of the hFMO2 gene is identified in Table 7A.

The previous comments apply as far as the specific comments on (a), (b), (c), (d) and (e) are concerned.

The invention also relates to fragments of these sequences, in particular sequences which encode polypeptides which have retained all or part of the activity of the FMO protein.

Some of these sequences may be identified by referring, in particular, to Table 7A, which provides an overview of the organization of hFMO2.

These partial sequences can be used for a large number of applications, as will be described below, in particular for making protein constructs of the FMO type or of different types, but also for producing, for example, FMO-like proteins.

Even if the sequences described are in general the normal sequences, the invention also relates to sequences which are mutated to the extent that they contain at least one point mutation, preferably mutations extending to no more than 10% of the molecule.

Preferably, the present invention relates to mutated nucleotide sequences in which the point mutations are not silent, that is to say they lead to a change in the encoded amino acid as compared with the normal sequence. Still more preferably, these mutations concern amino acids which form the structure of the FMO proteins or the corresponding fragments of these proteins, in particular in the regions corresponding to the catalytic sites, to the regulatory sites or to the sites for binding cofactors; the mutations may also concern the sequences which are involved in transport and targeting; they may also, in particular, delete cysteines or, on the contrary, make them appear, but also change the character of the protein either with regard to charge or with regard to hydrophobicity.

The present invention also relates to the mutations which may occur in the promoter and/or regulatory sequences of the human FMO genes, which mutations may exert effects on the expression of the protein, in particular on the level at which it is expressed.

In a general manner, the present invention is concerned with both normal FMO proteins and mutated FMO proteins as well as their fragments and the corresponding DNA and RNA sequences.

Among the nucleotide fragments which may be of interest, in particular for diagnosis, mention should also be made of the genomic intron sequences of the FMO gene, for example the junction sequences between the introns and the exons.

The invention encompasses the nucleotide sequences according to the invention which are distinguished in that they comprise at least the mutation G.1263mac.A, as will be defined below in the examples.

The invention also encompasses the nucleotide sequences according to the invention which are distinguished in that they contain at least 10 bases, as well as said nucleotide sequences, which can be used, in particular, as primers which are specific for an allele.

The invention also encompasses the nucleotide sequences according to the invention which can be used, in particular, as nucleic acid primers, which are preferably distinguished in that said sequences are selected from the sequences SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10.

The invention furthermore relates to the nucleotide sequences according to the invention which can be used, in particular, as probes which are specific for an allele and which are preferably distinguished in that said sequences are selected from the sequences SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13 and SEQ ID No. 14.

The invention also relates to the nucleotide sequences according to the invention which are distinguished in that said sequences encode one of the FMO domains.

The polypeptides which are encoded by the nucleotide sequences according to the invention, in particular the polypeptides having the sequence SEQ ID No. 3 or SEQ ID No. 6, naturally also belong to the invention.

In the present description, the terms protein, polypeptide or peptide are interchangeable.

The present invention relates to all the primers which can be deduced from the preceding nucleotide sequences and which can enable these sequences to be detected by using an amplification method such as the PCR method.

The present invention also relates to the nucleotide sequences which can contain unnatural nucleotides, in particular sulfur-containing nucleotides or nucleotides having an α or β structure.

Finally, the present invention naturally relates to both DNA and RNA sequences as well as to the sequences which hybridize with them and to the corresponding double-stranded DNA molecules.

Nucleic acid fragments of interest which should in particular be mentioned are anti-sense oligonucleotides, that is to say oligonucleotides whose structure ensures, by hybridization with the target sequence, that expression of the corresponding product is inhibited. It is also necessary to mention sense oligonucleotides which, by interacting with proteins which are involved in regulating expression of the corresponding product, induce either an inhibition or an activation of this expression.

As will be described below, it may be necessary, for some applications, to envisage mixed, protein/DNA/chemical compound, constructs, in particular the use of intercalating agents, for example; it should be understood that such compounds are covered by the patent as containing a sequence according to the invention.

The Proteins and Polypeptides

The present invention also relates to the proteins, polypeptides or peptides which correspond to the previously mentioned sequences and which are in unnatural form, that is to say that they are not used in their natural environment but that they were obtained by purification from natural sources or else obtained by genetic recombination, as will be described below.

The invention also relates to the same polypeptides or proteins which are obtained by chemical synthesis and which can contain unnatural amino acids.

The present invention relates to recombinant proteins which are thus obtained both in glycosylated form and in unglycosylated form and which may or may not possess the natural tertiary structure.

The Vectors and the Cells

The present invention also relates to cloning and/or expression vectors which contain a nucleotide sequence as described above.

These cloning and expression vectors can contain elements which ensure expression of the sequence in a host cell, in particular promoter sequences and regulatory sequences which are effective in said cell.

The vector in question can be an autonomously replicating vector or else a vector which is intended to ensure that the sequence is integrated into the chromosomes of the host cell.

In the case of autonomously replicating systems, which are prokaryotic or eukaryotic depending on the host cell, use is preferably made of plasmid systems or viral systems, with the viral vectors being able, in particular, to be adenoviruses (Perricaudet et al., 1992), retroviruses, poxviruses or herpesviruses (Epstein et al., 1992). The skilled person is acquainted with the technologies which can be used for each of these viruses.

Thus, it is known to use, as viral vectors, defective viruses which are cultured in complementing cells, thereby avoiding the possible risk of an infectious viral vector proliferating.

When it is desired to integrate the sequence into the chromosomes of the host cell, it is necessary to arrange for one or more sequences derived from the host cell to be integrated at each end of the nucleotide sequence in order to ensure that recombination takes place. The methods used in this case are also widely described in the prior art. Use can, for example, be made of plasmid or viral systems; examples of these viruses are retroviruses (Temin 1986) or AAVs, i.e. adenovirus associated viruses (Carter 1993).

The invention also relates to the prokaryotic or eukaryotic cells which are transformed with an above-described vector, with this transformation being to ensure expression of a natural or variant FMO protein or else, for example, one of its domains.

The animals which are distinguished in that they contain a transformed cell according to the invention also belong to the invention.

The invention furthermore encompasses a method for producing a polypeptide according to the invention, which method is distinguished in that a cell according to the invention is cultured and in that the protein which is produced is recovered.

As has been previously pointed out, the present invention also relates to the polypeptides which are obtained by culturing the cells which have been transformed in this way and recovering the polypeptide which is expressed, with it being possible to effect said recovery intracellularly or else extracellularly in the culture medium when the vector has been designed for ensuring the secretion of the polypeptide by means, for example, of a leader sequence, with the protein being expressed in the form of a preprotein or a preproprotein. The constructs which permit secretion of the polypeptides are known, both for prokaryotic systems and for eukaryotic systems. Within the context of the present invention, some of the FMO polypeptides may contain their own system for secretion or membrane insertion.

Preferably, the invention relates to the polypeptides which are specific for mutated forms of the proteins according to the invention, distinguished in that their sequences are selected from the polypeptide sequences which contain at least one mutation.

Cells which can be used for producing these polypeptides and which should be mentioned are, of course, bacterial cells (Olins and Lee, 1993), but also yeast cells (Buckholz, 1993), as well as animal cells, in particular cultures of mammalian cells (Edwards and Aruffo, 1993), but also insect cells in which it is possible to use methods employing baculoviruses, for example (Luckow, 1993).

The cells which are thus obtained can be used to prepare both natural or variant FMO polypeptides and also fragments of these polypeptides, in particular polypeptides which correspond to the different domains in question.

The invention also encompasses the monoclonal or polyclonal antibodies which are preferably directed against the polypeptides according to the invention, which antibodies are distinguished in that they are obtained by the immunological reaction of a human or animal organism with an immunogenic agent consisting of a polypeptide according to the invention, in particular a recombinant or synthetic polypeptide according to the invention; preferably, the immunogenic agent will consist of a polypeptide which is specific for the mutated form of the protein which is obtained in accordance with the previously described method, with the sequence of said polypeptide being selected from the polypeptide sequences which contain at least one mutation.

The invention also relates to the antibodies according to the invention, which are distinguished in that they are labeled antibodies, in particular for imagery.

These monoclonal or polyclonal antibodies, which are labeled and which correspond, in particular, to all or part of the mutated proteins, can be used, for example, in vivo or ex vivo as imagery agents on biological samples (imagery using antibodies which are coupled to a molecule which is detectable in imagery of the PET-scan type, for example).

The Cell Models

The transformed cells, as described above, can also be used as models in order to study the interactions between the FMOs and their partners, i.e. chemical and protein compounds which are directly or indirectly involved in FMO activity, and in order to study the different interactions which are involved depending on whether the FMO is a normal FMO or a variant FMO. However, in particular, they can be used for selecting products which interact with the normal or variant FMOs as agonists, in particular enzyme activators, or antagonists, in particular enzyme inhibitors.

Another potential application of the characterization of these genes is therefore the possibility of identifying compounds, in particular protein compounds, which interact with these FMOs. These compounds can be either inhibitors or activators, for example substrates or cofactors. Their identification makes it possible to use them in accordance with their interactions with the normal protein or the variant protein. In particular, it is possible to seek to isolate agents which have different effects on the normal FMOs and the variant FMOs.

It is also possible to use these cell models for studying the metabolism of xenobiotics, drugs or other compounds by a normal or variant FMO. This can be done in association with identifying the toxic potency of particular compounds, in association with selecting and developing compounds having reduced toxicity or having increased activity or in association with selecting and developing modified FMOs which have an increased ability to metabolize the compounds of interest.

This type of cell model can be constructed using genetic engineering techniques. Depending on the type of cell which it is desired to use, it is a matter of cloning the gene in question, in its normal form or in its mutated form, into an expression vector, whether it be an autonomously replicating vector or an integrating vector, with said vector containing all the elements for expressing the gene in the cell in question, or with the latter possessing all the elements for expressing the sequence in question.

This thereby results in eukaryotic or prokaryotic cells which are expressing the normal or variant FMO protein(s) and which can then constitute models for testing, at the same time, the interactions of different products with the FMO proteins or their variants or for testing compounds, in particular synthetic chemical products, which can interact with the product of the normal or mutated FMO gene, with these compounds being added to the culture medium of said cells.

It should, in particular, be noted that the products in question can equally well be agents having an antagonistic activity as agents having an agonist activity.

The use of cell models for the purpose of testing pharmaceutical compounds is well known, and once again there is no need to describe this type of model in detail. However, of the techniques employed, those which may be mentioned are phage display (Allen et al., 1995) and the two-hybrid methods (Luban and Goff., 1995).

These models can be of the in vitro type, for example cultures of human cells, either in normal culture or, possibly, in the form of an isolated organ.

The present invention also relates to organisms such as animals, in particular mice, which are expressing the phenotype corresponding to the normal or variant FMO of human origin. In this case too, these animals can be used as model animals for testing the efficacy of particular pharmaceutical products.

The present invention also relates to the products which are obtained by using the above-described cell models.

Diagnostic Method

As has previously been mentioned, the present invention relates, more particularly, to methods for diagnosing predisposition to FMO-linked disorders in a patient, which methods are distinguished in that a biological sample taken from said patient is used for determining the presence of a mutation in at least one sequence encoding an FMO by means of analyzing all or part of a nucleic acid sequence corresponding to said gene, with the presence of at least one such mutation being indicative of a predisposition of said patient to FMO-linked disorders.

It is important to make clear that, while the present invention only describes hFMO2 and hFMOx in detail, the diagnostic methods and the compositions for therapeutic purposes relate both to the abovementioned FMOs and to FMO1, FMO3, FMO4 and FMO5. This is because the FMOs, in general, are involved in the metabolism of xenobiotics and the disorders which are associated with them, such as, for example, the xenobiotics and the FMO-linked disorders which have been mentioned above.

The mutation, of those which have been investigated, which should be mentioned more specifically is the G.1263mac.A. mutation.

The analyzed nucleic acid sequences can equally well be genomic DNA, a cDNA or an MRNA.

While, as has previously been mentioned, the FMO-linked disorders which can be detected are more specifically understood as being the pathologies which are associated with xenobiotic metabolism, as mentioned above, or which are associated with the biological function of FMO, other disorders which could be linked to an FMO anomaly may also exist.

Although the diagnostic tools which are based on the present invention can make it possible to achieve a positive and differential diagnosis in a patient taken in isolation, they are preferably of value for achieving a presymptomatic diagnosis in a patient who is at risk, in particular with a familial case history, and it is also possible to envisage an antenatal diagnosis.

Furthermore, the detection of a specific mutation may enable a prognostic diagnosis to be made, in particular with regard to the intensity of the disorder or the probable time at which it will appear.

Of course, there are a very large number of methods for detecting the mutation in a gene as compared with the natural gene. These methods may essentially be divided into two broad categories; the first type of method is that in which the presence of a mutation is detected by comparing the mutated sequence with the corresponding natural, unmutated sequence, and the second type is that in which the presence of the mutation is detected indirectly, for example by detecting mispairings which are due to the presence of the mutation.

In the two cases, preference is given, in general, to the methods in which all or part of the sequence corresponding to an FMO is amplified prior to detecting the mutation, with these amplification methods being effected by means of so-called PCR or PCR-like methods. PCR-like is to be understood as referring to all the methods which employ direct or indirect reproductions of the nucleic acid sequences or else in which the labelling systems have been amplified; these techniques are, of course, well known; in general, they involve amplification of the DNA with a polymerase; when the original sample is an RNA, it is advisable first of all to carry out a reverse transcription. There are currently a very large number of methods for achieving this amplification, for example the methods termed NASBA “nucleic acid sequence based amplification” (Compton 1991), TAS “transcription based amplification system” (Guatelli et al., 1990), LCR “ligase chain reaction” (Landegren et al., 1988), “endo run amplification” (ERA), “cycling probe reaction” (CPR) and SDA “strand displacement amplification” (Walker et al., 1992), which methods are well known to the skilled person.

Table 1 depicts primer sequences which can be use for amplifying the sequences which are of interest in relation to the G.1263mac.A. mutation.

The reagent employed for detecting and/or identifying a mutation of the FMO gene in a biological sample comprises a so-called capture probe and/or a so-called detection probe, with at least one of these probes containing a previously described sequence according to the present invention.

Search for Point Mutations

In a general manner, several detection methods can be implemented, or adapted if necessary, after the sequences of interest have been amplified by PCR. The following may be mentioned by way of example:

1) Sequencing: comparing the sequences from several individuals and/or pinpointing a site of heterozygosity in a single individual.

2) “Single nucleotide primer extension” (Syvanen et al., 1990). Examples of primers which can be used for detecting the G.1263mac.A mutation by this method are given in Table 2.

3) RFLP “restriction fragment length polymorphism”. An example of a restriction enzyme which can be used for detecting the G.1263mac.A mutation by RFLP is given in Table 3.

4) Searching for “single strand conformation polymorphisms” (SSCP).

5) Methods based on cleaving the mispaired regions (enzymic cleavage with S1 nuclease, chemical cleavage with different compounds such as piperidine or osmium tetroxide, etc.

6) Detecting a heteroduplex by electrophoresis.

7) Methods based on using allele-specific oligonucleotide probes in hybridization: “allele specific oligonucleotide” (ASO) (Stoneking et al., 1991). Examples of probes which can be used for detecting the G.1263mac.A mutation by ASO are given in Table 4.

8) OLA “dual color oligonucleotide ligation assay” method (Samiotaki et al., 1994).

9) ARMS “amplification refractory mutation system” method or ASA “allele specific amplification” method, or PASA “PCR amplification of specific allele” method (Wu et al., 1989).

This list is not exhaustive and other well known methods may also be used.

Searching for Alterations, for Example of the Deletion Type

Other methods which are well known and which are based on hybridization techniques using genomic probes, cDNA probes, oligonucleotide probes or riboprobes may also be used for searching for this type of alteration.

The methods, according to the invention, for diagnosing a predisposition to FMO-linked disorders in a patient, which are distinguished in that said analysis is carried out by hybridization, with said hybridization preferably being performed using at least one oligonucleotide probe which is specific for the allele, or in that the presence of a mutation is detected by comparison with the corresponding natural, unmutated sequence, or in that said analysis is carried out by sequencing or by electrophoretic migration, more specifically by SSCP or DGGE, or in that said analysis is performed using a methodology which is aimed at detecting a truncation of the protein, therefore also form part of the invention.

The methods, according to the invention, for diagnosing a predisposition to FMO-linked disorders in a patient which are distinguished in that all or part of the nucleic acid sequence of the FMO gene is amplified prior to detecting the mutation(s), with the amplification preferably being performed by PCR or a PCR-like method, and the primers selected for performing the amplification preferably being selected from the primers according to the invention, also form part of the invention.

The reagents for detecting and/or identifying a mutation of the FMO gene in a biological sample, which reagents are distinguished in that they comprise a so-called capture probe and/or a so-called detection probe, with at least one of these probes containing a sequence according to the invention or an antibody according to the invention, also form part of the invention.

Methods which are Based on Detecting the Gene Product

The mutations of the FMO gene can be responsible for different modifications of the product of this gene, with it being possible to use these modifications for a diagnostic approach. Thus, the modifications in antigenicity can make it possible to develop specific antibodies. All these modifications can be used for the purpose of a diagnostic approach due to the existence of several well known methods, such as the RIA method or the ELISA method, which are based on using monoclonal or polyclonal antibodies which recognize the normal protein or mutated variants.

Finally, it is also possible to diagnose a predisposition to FMO-linked disorders in a patient by measuring the enzyme activity of the FMO(s) in biological samples taken from said patient. Thus, measurement of this (these) activity(ies) can indicate, when compared with an internal or external standard, a predisposition to one of the abovementioned disorders.

Therapeutic Compositions

The present invention also relates to curative or preventive therapeutic treatments of FMO-linked disorders.

Use can be made of the compounds which are directly or indirectly involved in FMO activity and which are derived from using the previously described cell models.

Use can, in particular, be made of the compounds which are able to interact, in particular as agonists or antagonists, with the normal or variant FMOs.

The present invention also relates to therapeutic compositions which comprise, as the active principle, a compound which is able to modulate FMO activity; these compounds may be compounds which have a pro-FMO activity, in particular as previously described, or compounds which have an anti-FMO activity.

In a general manner, a compound which has a “pro-FMO activity” is understood as being a compound which induces FMO activity, in contrast to an anti-FMO compound, which has a tendency to reduce FMO activity. The actual effect of these types of activities will depend on the type of enzyme, i.e. normal or pathological, which is expressed.

Preference is given to using therapeutic compositions whose activity differs toward normal FMO enzymes and variant FMO enzymes.

It is first of all possible to envisage a substitution treatment, that is to say therapeutic compositions which are distinguished in that they comprise, as the active principle, a compound having a pro-FMO activity; these compounds can, in particular, be all or part of polypeptides as have previously been described or else a vector for expressing these same polypeptides or yet again chemical or biological compounds which possess a pro-FMO activity or an FMO-like activity or which induce production of FMO.

It is also possible to use therapeutic compositions in which the active principle has an anti-FMO action, in particular an anti-FMO variant action. In this case, the treatment is a suppressive treatment. The compounds can, for example, be compounds which interact with said enzymes, in particular protein compounds, in particular anti-FMO antibodies, in particular when these antibodies recognize the variant proteins. The compounds can also be chemical products which possess an anti-FMO activity, in particular antagonists of variant FMO.

Of the large number of pharmaceutical compounds which can be used, those which should more specifically be mentioned are the anti-sense sequences which interact with the normal or mutated FMO gene, or else the sense sequences which act on the regulation of the expression of these genes, with said products being able to interact downstream of the expression products which are induced by the FMOs.

The monoclonal antibodies which inhibit the FMOs, in particular the mutated FMOs, and/or which inhibit the corresponding ligands and/or the products which are induced by FMO activity, and which can, therefore, have pro or antiactivities, should also be mentioned.

It is also possible to envisage expressing proteins, or their fragments, in vivo, in particular by means of gene therapy, using the vectors which have been previously described.

Within the context of gene therapy, it is also possible to envisage using the “naked” sequences of the previously described genes or cDNAs, with this technique having been developed, in particular, by the company Vical, which demonstrated that it was possible, under these conditions, to express the protein in particular tissues without resorting to the support of a viral vector, in particular.

Still within the context of gene therapy, it is also possible to envisage using cells which are transformed ex vivo, which cells can then be reimplanted either as such or within systems of the organoid type, as is also known in the state of the art (Danos et al., 1993). It is also possible to envisage using agents which facilitate the targeting of a defined cell type, penetration into the cells or transport toward the nucleus.

Thus, the invention also relates to a therapeutic composition which is distinguished in that it comprises, as the active principle, at least one compound which is able to modulate FMO activity, preferably FMO2 and/or FMOx activity.

The invention also encompasses a therapeutic composition which is distinguished in that it comprises, as the active principle, at least one compound which is able to interact with FMO and preferably able to interact with FMO2 and/or FMOx, or a therapeutic composition according to the invention which is distinguished in that it exhibits different activities on normal FMO and on pathological FMO.

The invention also encompasses a therapeutic composition according to the invention which is distinguished in that it comprises, as the active principle, a compound having pro-FMO activity, which compound is preferably selected from the following compounds:

a) a protein or a polypeptide according to the invention,

b) an expression vector according to the invention,

c) a nucleotide sequence according to the invention, distinguished in that said sequence is a sense sequence which induces FMO expression.

The invention furthermore relates to a therapeutic composition according to the invention which is distinguished in that it comprises, as the active principle, a compound having an anti-FMO activity according to the invention; the active principle is preferably selected from the following compounds:

a) an anti-FMO antibody according to the invention,

b) an expression vector according to the invention,

c) a nucleotide sequence according to the invention, distinguished in that said sequence is an antisense sequence which inhibits FMO expression,

d) a nucleotide sequence according to the invention, distinguished in that said sequence is a sense sequence which inhibits FMO expression.

The invention also relates to a therapeutic composition according to the invention, which composition is distinguished in that the active principle is a soluble sequence which interacts with FMO.

The invention also relates to the use of an active principle, preferably at least one product according to the invention which is able to modulate or interact with FMO, FMO2 and/or FMOx, for producing a drug which is intended for treating and/or preventing disorders which are linked to FMO function.

Under another aspect, the invention relates to a method for biodegrading or biosynthesizing an organic or inorganic compound, which method is distinguished in that it employs a polypeptide or a cell according to the invention.

Thus, the polypeptides having an FMO activity according to the invention can advantageously be used for biodegrading, in accordance with the oxidation reactions as described, for example, by Ziegler (Ziegler et al., 1993), the compounds which are FMO substrates, in particular the compounds as mentioned in the present description, or be used for biosynthesizing a compound of interest from said compounds which are FMO substrates, in particular for biosynthesizing a drug, a food additive, a pesticide or a herbicide.

The methods for elaborating a compound of interest, which methods are distinguished in that they use a polypeptide or a cell according to the invention do of course form part of the invention. Thus, the polypeptides or cells according to the invention can advantageously be used in vitro for determining the potential metabolism of the compound of interest and for analyzing the metabolites which may possibly be obtained, including their toxicity and/or their activity. The results which are obtained make it possible to confirm the compound or to reformulate it such that it does or does not become an FMO substrate or such that the metabolites which are formed are different.

The products which can be obtained using said biosynthetic method also form part of the invention.

Finally, the invention encompasses the use of a polypeptide or a cell according to the invention for detoxifying a xenobiotic compound which is an FMO substrate. These xenobiotic compounds can be present in the environment, as a pesticide or a herbicide, be present naturally in plants, as particular alkaloids, or can correspond to pharmaceutical compounds.

Taking into account the homologies of the known messenger RNAs of genes of the flavin monooxygenase family, these genes share the same exon/intron structure:

exon1: untranslated, variable in size and sequence,

exon2: beginning of the coding region, encodes amino acids 1-44,

exon3 : amino acids 45-107,

exon4 : amino acids 108-161,

exon5 : amino acids 162-209,

exon6 : amino acids 210-275,

exon7 : amino acids 276-394,

exon8 : amino acids 395-419,

exon9 amino acids 420-535, end of the coding region and 3′ untranslated region.

The introns vary in size and complexity. We firstly isolated the sequence of three fragments from BAC 123H04M, which fragments contain all the exons of this homologue.

Fragment 1 : containing exons 1 and 2,

Fragment 2 : containing exon 3,

Fragment 3 : containing exons 4 to 9.

The sequences of two introns were then completed and the structure is depicted in Table 7.

Isolating BAC 123H04M

A BAC (“bacterial artificial chromosome”) which corresponded to the candidate region which had previously been located on chromosome 1, was isolated in order to identify a gene encoding a novel FMO. A library of BACs covering the complete human genome was prepared from the DNA of a human lymphoblast cell line which was derived from individual No. 8445 of the CEPH families. This cell line was used as the source of high molecular weight DNA. The DNA was partially digested with the restriction enzyme BamH1 and then cloned into the BamH1 site of the plasmid pBeloBacII. The resulting clones were pooled and screened using a three-dimensional analytical procedure which had previously been described for screening libraries of YACs (“yeast artificial chromosome”) (Chumakov et al., 1992). The three-dimensional pools which were obtained were screened by PCR using primers which flanked the D1S3423(WI-10286) marker. This STS (“sequence tagged site”) had previously been located in the candidate region. One clone, of BAC 123H04M, was thus isolated.

Following digestion with the restriction enzyme NotI, the size of the insert carried by this BAC was determined in an 0.8% agarose gel after electrophoretic migration in an alternating field (CHEF) (4 hours at 9 volts/cm, with an angle of 100°, at 11° C. in 0.5×TAE buffer). This demonstrated that BAC 123H04M carries an insert of 180 kb.

Determining the Chromosomal Location of BAC 123H04M by Fluorescent in-situ Hybridization (FISH)

The chromosomal location of the BAC in the candidate region 1q23-q25 was confirmed by carrying out fluorescent in-situ hybridization (FISH) on metaphase chromosomes using the method described by Cherif et al., 1990. More precisely, BAC 123H04M was found to be located in band 1q23 of chromosome 1.

Sequencing the BAC 123H04M Insert

In order to sequence the BAC 123H04M insert, three separate libraries of subclones were prepared from the sonicated DNA of this BAC.

After incubation overnight, the cells derived from three liters of culture were treated by alkaline lysis in accordance with standard techniques. After centrifuging the resulting product on a cesium chloride gradient, 52 μg of the BAC 123H04M DNA were purified. 7 μg of DNA were sonicated under three different conditions in order to obtain fragments whose sizes were distributed uniformly over the range 1 to 9 kb. The resulting fragments were treated, in a volume of 50 μl, with 2 units of Vent polymerase at 70° C. for 20 minutes in the presence of the 4 deoxytriphosphates (100 μM). The blunt-ended fragments which resulted from this step were separated by electrophoresis in a 1% low melting point agarose gel (60 volts for 3 hours). The fragments, which were grouped according to their sizes, were excised and the bands which were obtained were treated with agarase. After extraction with chloroform and dialysis on microconcentrators trademarked as Microcon 100 columns, the dissolved DNA was adjusted to a concentration of 100 ng/μl. A ligation, involving overnight incubation, was performed by bringing 100 ng of the fragmented BAC 123H04M DNA into contact with 20 ng of the vector DNA, which had been linearized by enzymic digestion and treated with alkaline phosphatase. This reaction was carried out in a final volume of 10 μl and in the presence of 40 units of T4 DNA ligase (Epicentre)/μl. The ligation products were then used to transform, by electroporation, either an XL-Blue strain (for multicopy plasmids) or a D10HB strain (for the subclones derived from the BAC). The clones which were lacZ − and resistant to the antibiotic were repicked individually into microplates for storage and sequencing.

This resulted in:

864 subclones derived from the insertion of fragments of from 2 to 3 kb in size into the SmaI site of plasmid puc18;

1728 subclones corresponding to the insertion of fragments of from 1.5 to 2 kb in size into the BamHI site (rendered blunt) of the plasmid trademarked as BluescriptSK;

288 subclones carrying fragments of from 4 to 7 kb in size which were inserted into the PmlI site of a modified BAC vector.

The inserts of these subclones were amplified by PCR, which was carried out on bacterial cultures which were incubated overnight and which used the vector primers which flanked the insertions. The sequences of the ends of these inserts (on average 500 bases at each end) were determined by automated fluorescent sequencing on an ABI 377 sequencer which was equipped with the ABI prism DNA Sequencing Analysis package (version 2.1.2).

The sequence fragments derived from the subBACs were assembled using R. Staden's Gap4 package (Bonfield et al., 1995). This package enables a complete sequence to be reconstructed from sequence fragments. The sequence deduced from aligning the different fragments is the consensus sequence.

Finally, directed sequencing techniques (systematic primer progression) were used to perfect the sequences and link the contigs.

Analysis of the Sequences

The potential exons of BAC 123H04M were pinpointed by carrying out homology searches on the public protein, nucleic acid and EST (expressed sequence tags) databases.

Databases

Use was made of local revisions of the main public databases. The protein database employed consists of the non-redundant fusion of the Genpept (automated GenBank™ translation, NCBI; Benson et al., 1996); Swissprot (George et al., 1996); and PIR/NBRF (Bairoch et al., 1996) databases. The duplicates were eliminated using the “nrdb” package (public domain, NCBI; Benson et al., 1996). The internal repetitions were then masked with the “xnu” package (public domain, NCBI; Benson et al., 1996). The resulting database, designated NRPU (non-redundant protein unique) was used as a reference for the protein homology searches. The homologies which were found with this database made it possible to locate regions which potentially encoded a protein fragment which was at least related to a known protein (coding exons). The EST database employed is composed of “gbest” subsections (1-9) of Genbank (NCBI; Benson et al., 1996). It contains all the public transcript fragments.

The homologies which were found using this database made it possible to locate potentially transcribed regions (present on the messenger RNA).

The database of nucleic acids (other than the ESTs) which was employed contains all the other subsections of Genbank and EMBL (Rodriguez-Tome et al., 1996), the duplicates of which were eliminated as described above.

Packages

Use was made of all the BLAST package (public domain, Altschul et al., 1990) for searching for homologies between a sequence and protein or nucleic acid databases. The significance thresholds depend on the length and complexity of the region tested as well as the size of the reference database. They were adjusted and adapted for each analysis.

Identification of FMO-Associated Genetic Polymorphisms in Relation to a Phenotypic Polymorphism which is Associated with the Occurrence of Juvenile Glaucoma, J-POAG, which is a Disease which is Transmitted in an Autosomal Dominant Manner (Locus GLC1A)

Detection of polymorphisms/mutations

1) Extracting the DNA

The DNA is extracted from the peripheral venous blood following cell lysis, protein digestion, organic partition and, finally, precipitation with alcohol.

The blood (20 ml) is drawn, by peripheral venous puncture, into a tube containing EDTA.

It is diluted with an equal volume of double distilled water. After 10 minutes, the cells are collected by centrifuging at 1600 g for 10 minutes. This manipulation is repeated.

The white cells are lysed in the presence of 20 ml of CLB buffer (10 mM Tris, pH 7.6, 5 mM MgCl 2 , 0.32 M sucrose, 1% (v/v) Triton X-100). The nuclei are collected by centrifuging at 1600 g for 10 minutes. This manipulation is repeated.

The nuclei are washed once in RSB buffer (10 mM Tris, pH8, 10 mM NaCl, 10 mM EDTA). The pellet is resuspended in 2 ml of RSB buffer to which sodium lauryl sulfate (1%) and proteinase K (200 mg/ml) are added. The mixture is incubated at 55° C. for at least 3 hours and shaken regularly.

The resulting DNA solution is then extracted with one volume of phenol which is equilibrated with a 50 mM Tris, pH 8, buffer. This operation is repeated and finished off with an extraction with one volume of chloroform/isoamyl alcohol (24:1 v/v).

The DNA is precipitated with one volume of isopropanol, rinsed with ethanol (70%), dried and finally resuspended in 1 ml of TE buffer (10 mM Tris, pH 8, 0.5 mM EDTA). The concentration of DNA is determined by measuring the absorbance at 260 nm and taking 50 μg/ml of DNA as being equivalent to one absorbance unit. The DNA concentration is then adjusted to 200 μg/ml.

2) Amplification of the Genomic DNA

The oligonucleotide primers employed for the genomic amplification of the BAC 123H04M-derived exon sequences, as predicted by computer analysis, were defined using the OSP package (Hillier et al., 1991).

All these primers contain, upstream of the bases which are specifically targeted by the amplification, a common oligonucleotide tail which is intended to enable the amplified fragments to be sequenced (PU for the upstream primers and RP for the downstream primers; sequences shown in Table 5).

The oligonucleotide primers were synthesized on a GENSET UFPS 24.1 synthesizer using the phosphoramidite method.

Each predicted exon sequence was amplified by polymerase chain amplification reaction (PCR) under the following conditions:

The amplification is performed in a Perkin Elmer 9600 or MJ Research PTC200 thermocycler with a heating lid. After heating at 94° C. for 10 minutes, 35 cycles are carried out. Each cycle comprises: 30 seconds at 94° C., 1 minute at 55° C. and 30 seconds at 72° C. A final segment of elongation at 72° C. for 7 minutes terminates the amplification.

The quantity of amplification products obtained is determined by fluorometry on a 96-well microplate using the intercalating agent Picogreen (molecular probes).

3) Detecting Polymorphisms/Mutations

Sequencing

The products of the PCR genomic amplification were sequenced on an automated ABI 377 sequencer using fluorescent primers, which were labeled with the ABI fluorochromes (Joe, Fam, Rox and Tamra), and Thermosequanase DNA polymerase (Amersham).

The reactions were performed in 96-well microplates on a Perkin Elmer 9600 thermocycler under standard temperature cycle conditions:

8 cycles: denaturation: 5 sec. at 94° C.; hybridization: 10 sec. ; elongation: 30 sec. at 72° C., then

13 cycles: denaturation: 5 sec. at 94° C.; elonga-tion: 30 sec. at 72° C.

6 units of Thermosequanase and 5-25 ng of amplification product were used per sequencing reaction.

Once the amplification cycles have been completed, the sequencing reaction products are precipitated in ethanol, resuspended in a loading buffer containing formamide, denatured and deposited on 4% acrylamide gels; the electrophoreses (2 hours 30 min at 3000 volts) are conducted on ABI 377 sequencers which are equipped with ABI collection and analysis software (ABI Prism DNA Sequencing Analysis Software, version 2.1.2.).

Analyzing the sequences

Since J-POAG is an autosomal dominant disease, the sequence data obtained were analyzed in order to detect the presence of heterozygosity sites in the patients suffering from juvenile glaucoma. The heterozygosity sites were confirmed after comparing the sequences of the two strands of genomic DNA from each individual concerned. A heterozygosity site is selected as a candidate mutation responsible for the occurrence of FMO-linked disorders when it is present in a population of members of one and the same family while being generally absent from the controls who are not related to the family.

Results

Out of all the BAC 123H04M-derived amplification fragments studied, one exhibits a heterozygosity site which segregates with the occurrence of juvenile glaucoma in a pedigree depicted in FIG. 1 .

This heterozygosity site (G/A) is present in 7 patients suffering from J-POAG whereas it is absent from 3 healthy homozygous patients (GIG), with all the patients being derived from the same family. Furthermore, 99 unrelated controls are similarly homozygous (G/G) for this site, indicating that the frequency of the A allele in the general population is less than 0.005.

The site is contained in exon 8 of the gene which encodes the hFMO2 protein according to the invention; the described mutation transforms glutamic acid in position 402 of the sequence SEQ ID No. 1 of hFMO2 into lysine.

It is surprising to note that calculating the lod scores which integrate the preceding data for different assumptions of the frequency of each allele in the general population indicates a probability of greater than 100 to 1 that the described heterozygosity (G/A) is linked to J-POAG (Table 6). This probability is significant due to the fact that the analysis related to one single family.

The primers which enabled the DNA fragment containing this heterozygosity site to be amplified are described in Table 1.

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14

1

26016

Homo sapiens

genomic

1

catcagttat ccctggagga taactaagcc atctgcctcc atcatctttt aagggttcag 60

tcagtttaaa actttgcttc tatacctagg tattttcttt tctctgtatg ttggtcaggt 120

acaattattt ttaacagggc ttccatcaat atcataacta cctagagaag acattgcaaa 180

gataaaattg gagaattgtt aacaggctgt taacaaaatg tgtacccaac tgccaatgaa 240

gtggcttgat ttttttcttt ttttaaaatt tttcttttgt atccttttat tttatttact 300

tattttttag agacacagtc tcgctctgtt acccaggctg gcgtacaatg gcacaatcat 360

agcccactgc agtctcgacc tccagggcct caagcaatcc tcccacctca gccttccaag 420

tagctgagac tacaagtgca tgctgccatg cctgactgat tttttgtttt ttgcagagat 480

gaagtctcac tatgttgccc agtctgacct tgaactccta gcaataccct accctggcct 540

cccaaactgc tgggattaca ggcatgagcc actgtgcctg gccttttttc attttaactg 600

agaaatgtgt tcagctcttt tgttccttag tcattgatca tcacttttgt tatatctgtt 660

agtcttgtca tagagttgct gcacttatta cacagagaag gccttttatc acgaccaatt 720

tattttagga aatttcaggg aaaacgtttt tctagaacac cttatttgac attataaaac 780

aactcttcac tcttgcactc cagacctccc tttccagttt tctttttctc catagtggtc 840

atcaccactt gttttatttt attgatgggc tgtctggctc cctcaactgc aaagtaaact 900

ccacaaaggc agagagtttt gtctctttta ttcattgctg tacctgcatc acttagaaag 960

tttctggcac ctaggaagtg ttcagtaaat atttattgaa taagtttatg taaaacgtct 1020

cagactcctt agagaaactg gtcttttggg gttggagaat aaagttcttt acctcatcag 1080

ttagactcta tctaaggtac acgagggctt gctagtctcc taagttagtc tgctaataaa 1140

tgttaaccct aataactgaa attattagca gaggtaatta tccagttcta tatcaaggca 1200

aaaagacagc agtggataga aagatcttag aagtcccact aggttcatcc aagccaccat 1260

acacataggc agaaaaatca aaataagata tgagcctgga cagggtgagc aatctgggaa 1320

aagatgaaca cagtatgcta ggacccagaa atcatcaagt ctatgaaaac taagccagaa 1380

cacaaatgtg aattccataa gatcaggaac ataatctgtc ttgttcatcc aggcatggta 1440

atctgccaga aatagtgctt aactgcaaga actgaatatt tgttagataa ttaaaccatc 1500

aactaaatga gattcatgca accatgaaaa atgctgctat aggtacacaa tattgatata 1560

ctagaaagtt aaaaaatcaa gttggaaatt agactattcc atttctgttt gtgtgtatgt 1620

atctacaaat aggtggaagg atataccaaa atgtcaacag cagttacctc tgggtggtga 1680

ggagtaatct taaccttgtt atttatccct atatgttcat ttgtgaatga atatttatta 1740

catcattata aaaaggattt ttaaactatc tgtatgttta agagtatatg ttgctactat 1800

gtaagagtat atgctgttac tgtaaagaca ttgcattact actgttgacc tcagagcacg 1860

cgcctcttgc ctaattctag gactcctaac taagtctttg gagtttcagc tggaagaatg 1920

ctggaggaat acggaactcc tcccatttct cacagccacc tccaactctt aaaaacgctt 1980

ccaactgcct cccagcacac aaccaaggga gaaaactatt ctgtcaaaga gacggtgcca 2040

aaaggcaaaa acaaaggtaa ggatgatcgc tggggaaaga agctgaaaag gaaaagctca 2100

gaactctagc tggaaatttg gctcacatcc ctagtatgtt actgcatagt ctggctttgt 2160

tcaatgggtc gcttttaaat attaaagcta gatgtaagca aggtttgcaa caaagtccat 2220

aagaaactca gcttttctca aaggcaagaa gagagcagga tttttgactg gctctttatt 2280

caatagtgct gcttattaaa ttaccactgc tacaatgttt aaagccaatt acctgagcac 2340

atcataagga ttctcttacc ggttgtccca gttaagtaat gttgattgat caactccttg 2400

acaggagctg atggcaaaga aggtagctgt gattggagct ggggtcagtg gcctaatttc 2460

tctgaagtgc tgtgtggatg agggacttga gcccacttgc tttgagagaa ctgaagatat 2520

tggaggagtg tggaggttca aagtaagtga gattttcttg ggtcttgaac aggttgtgtt 2580

gttatttcag ggtgaatcac agttactgat gggtcatatt gagaaattta ttaaacaact 2640

ctgatcagat tttatttcta tttattgatg tggccataat ggaactgaag tcataggctg 2700

gcatctctcc cccagtcaat actaacccaa cccaggtagc tgacccaggc atgtaaaaga 2760

tctcttcttt tggattcagc aattgtctta cagcccatac ttctgtcatt ctttaatacg 2820

ctaatattag agaacatttt acaaaaatag aagtaacagg gattcttctc aagatatcac 2880

ttctgtttca attattaaac caaatgcttc tttagagacc atgctcttat cattactatt 2940

tttctctgac aaatgaagca tgtttgttta ctgagcttta tcaatgacat tctagtataa 3000

ctgctgtgaa actctttgtt aaatatgttt tattaaattt attctattaa tcaaaccaaa 3060

atattgataa tgctatttgt ctgtattagt ccattctcat gctgctatga agaaatactg 3120

agactgggtg atttataaag gaaagaggtt taattgactc ccagttccac aatgctgggg 3180

aggactcagg aaatttacaa tcatggcagt gggaaagaga ggtgctgagc aaagggggaa 3240

aagcccctta taaaaccatc agatctcatt agaacgcact cactatcatg agaacagcat 3300

gagggtagct gcccccttga ttcaattacc tacccccacc aggtccttcc caagacatgt 3360

ggggattgtg ggaactacaa ttcaatatga gatttggatg gggacacaaa gctaaaccat 3420

gtcactgtcc ttaaaaattt gtataaaact tagaaagttg catagatagc tataaggagt 3480

tacaattatt ccttcccaca acctctcaat aggtagtagc ttaccacctt ctagctgtga 3540

gatcttgagc aagttattta catcctgtgt ttcaatttac tcagttataa atggatataa 3600

taacaggaaa gtgtgattat ctcatagtgc tattttgaag attaagggag ataattcata 3660

taaagaactt agataagttc cggactcata gagttcaata aatgttagct actaataata 3720

actatatatt ttatagatga gcaaactgaa agtgagggag gttaagtgag atggccaggg 3780

ccacacaact ggaggaactg gccttcaaac cacggcctac gtgacttcta aacagataag 3840

ccctgactta caaccatgcc ctaacttgca ttcttgctca aaaagattaa acaaaagttt 3900

aagttcagaa cccaaaagca atgactttag aattatgtaa tcaggtatcc ctgagatatt 3960

aaaacacata agaatattcc aaatgggagc aaaaggtttg aatacatgaa aatcaaactc 4020

atatcagcag agaccatata aagggctctc actgcaggct gactagttag gaggatggca 4080

aggtgatcca ggacctgcgc atgctttgtc agttcaaatt gaatctcatg ccaacagcga 4140

tcttttttaa catgtaacat taggtgtctc aggtacacat gaccataaac cacacctgga 4200

gggtttcttt tattttcttt ttaatatttt tctgagacag ggtcctactc tgtcacccag 4260

gctaccatgc ccagccatgg agagtttctt aaagatactg attcctttgg ttaaacctgc 4320

caccaaaaaa aaaaaaaaaa aaaaaaaaaa atactgattt gtgggcactc catcccaaat 4380

ctatggaatc aaaatcttct gggggttttt aataaacatc tcaaatgaat cctatgataa 4440

gacaaatttg gtaattgtta cacaaacacc taatttaaaa atctgatcat tctactatct 4500

aaacacactc agagttaatg agggagaagg gagaaattga ttcttctgta agacaggtag 4560

ctttgcaaaa aggaaaacag cttaaatcac attcatttct tattaaaagc tgatgattaa 4620

tatcatttta gtttttcctg ggatggtgat ataatatggt ggtcattcct gtcttaacca 4680

aagatatttt tgtccactct aggttcacat gtagatttca gctggaattt tttttttttt 4740

ttttttttgc tcccaggtag attcttaacc taaacaagaa atgtagaaat tacagttggt 4800

ccttggtata tgcaggagat tggctccaca acctccctcc cccagtatac caaaatcctt 4860

gcatactcac atcccacaga tttattgtca gcaaaagaga tgagagttag tttgaacagt 4920

ctgccaacaa tatgatttga tgaattctag gaaggtattt tctgcagtaa aatatttctc 4980

caactatcct tttgccagta tctaaaattt cagattagag ataacttcct attcactaga 5040

aaaactggat taaaacctga ttaattaggc tttattgaat attaagggtt aagtatataa 5100

ctgtggaact tgtaacagta tcacatttca aatttctctt aaaactatat ccaatagagg 5160

aatgtaaact attgtctcca ctcaacgaag tcaaagagtc caaagagtct ccctgcagag 5220

tgaaacataa aataagcaaa atttcatagg ctgcctgcac tacggctatg tgagggtttt 5280

ggttaccagg tgactgggag tttccaagaa ggatgctggg agccccatgc tcttccctgg 5340

gaaactttgc cttttcacta ctctaccatc cagaagcaat tttttaaatg ggttatttat 5400

taattttcgt atttacacaa ctcctactga gattacttaa catatttggt ggtgacaagt 5460

taacaataaa taagtaaatt taagaatcct tgtcctatac ccaacccaga caatagagtt 5520

cttccagact ctccagcacc ccctagtggc acatatggac catgggacgg gtaggtaatt 5580

agcatatatt tttcgttctg tttccagcaa cgggaagcac ttggcaagca tcaccttctt 5640

ttcttcgcaa tactgctagg aagtatgtat tatgattatc tttatttaca tattaagaag 5700

aaacagtttt cagataaaga atttgctcag gggaacatag gtggcgggag aaaaaaaacg 5760

agggtttaca atttcggagc tctcacactt aataaccttg ctgaagtatt gatagaggaa 5820

aacatgatct tctttcagcc gctaaccttc tctgtttcct ttattgttcc taataccttg 5880

tattcacgtg ggagttacca tgtacatttt ttttcctgtg ggttttcttt taatatttgg 5940

atttggatct cctccttttc cagatgtata tgtttagtta ttttaatttt catgtaatac 6000

tctctagaca tatctcaatc ttggttttct tcctctaagt tcaatctgaa atatcacttt 6060

ctctcttaaa tttggctccc ccaagatcca acattccaaa catattgcca atgagtgtat 6120

accttttagc ttgaaagcag cagaaaaaaa gtggtaaata cctgagccag ggaacttaat 6180

tagggggttc tatcagtgat caaggccagt gatcaaggga gacaccagcc taatgaaaga 6240

tgacagaaga tagcaatact ctaatagaga tgtggttcac aaagttcatt gtgcagaagc 6300

agctagggag agcttctaaa atacagaaat ctgagcccgt cttttttctt ttcttttttt 6360

tttttttttt tttgagatgg agtctcgctc tgttgcccag gctggagggc agtagcgcaa 6420

tctcagctca ctgcaacctc catcccccgg gttcaagcga tttagctggg attacagcct 6480

tgtgccacca cacatctgga cccatcttct aatgcaactg gtccactgac tggcatttgg 6540

gaattgcaat tttgcctcta attgtaggac aaggaagtaa gaagagtttt aatcatattc 6600

aattcaagta atggagcaga tagatgtaag gtccatccga aagagtgaaa tgatagaatc 6660

acagaatatt cttaaagaaa ggcaatttta ttctttctaa ctgcttatgg taactaccca 6720

tgaaagcaaa aatattgatt ggtaagggtc aatataatga tgtttcacga agaaaaagtt 6780

taatttgtaa gtttttgtaa ttcacattta taataaataa atctgtttct gctttataaa 6840

tttcctcact tgagtagatt aaatattacc cttataatct tctttaaact tactgtttac 6900

aaccttttta ttgtcatgaa gtcaaacata aacttcaatt cagctcgtga tcaaaagatc 6960

ataaattcta aataagtgct atctgaatta acttggtttg ctagagtttt ctgacattct 7020

gaaaattcta tattagaaga attctttatt atatgataat ttatgttaaa caaattatag 7080

caaattctac acataaggaa attcagacta tatttatgct taattatcca ggcagtagta 7140

gtacttaagt aaatatgtga gttaaattta tctgttttga aaactgtgcc tctgtcctcc 7200

tcttgattga caataaaccc tctgtctcca ctttcacatc tccaaagttc aagtgcattt 7260

taatacaata taacaataag caccataaag atataaacta tgtttgtact gttagcatct 7320

tatccctaaa tccaagctca ggccctggtc agttcaagca tttgatacat acttgtctat 7380

taaatcaaca ttaatcatct cttcataact aggaaaacta ggccaatttt acccagattt 7440

gtctaaatac acagatgcct acttcagcaa actaaatgta gaaggaagca catatgaaga 7500

caagggggtc ttttttagct gctatttacc aattaaccca acaataaaag tttatcactt 7560

ggctgggcgc ggtggctcac gcctgtaatc ccagcacttt gggaggctga ggcaggtgga 7620

tcacctgagg tcgggagttc gagaccagcc tgaccaaaat ggagaaaccc caactctact 7680

aaaaatacaa aattagccgg gcatggtggc gcatacctgt aatcccagct actcaggagg 7740

ctgaggcagg agaatcactt gaacccaaga cggggaggtt gcagtgagcc gagatcatga 7800

cattgcactc cagcctgggc aacaagagca aaattctgtc tcaaaaaaaa aaaagggatt 7860

atcacttgat cttcagaaaa atagtgaggt cattattgtt tgctgacaga ctacacaagt 7920

aaaatctccc aaaggccagt tttgccctgg ccctaagatt actgtagggc ctcagacatc 7980

aaatcagttc ttctcatcac tcaaaattcc cttaaaattg acctgacaga gaagccaacc 8040

acatttttaa gccaaattgt tgggtctttt aaaaactagc attttggctg tagtataaca 8100

gtcttagttt aactgattca aaactatggc tggcttagta aatttaacgc tagtggccaa 8160

taataacaga aaagagataa atattcttaa gtatgtattt tgagccaggg attctgctaa 8220

gtactttatt cactctcatt aaagccttga aacaattgtt gcatgtttaa gttattaatg 8280

agccccattt tacagaggaa aatgaggaaa ctgacctatg taacttgctc atggtcacaa 8340

gccattaaag gtggcagaat taggatatca atccagtcgg tgtgactcca gaaccctcct 8400

atttactcta tactactcat aaaattattt ggtcttgggg ctgggcgcag tggctcatac 8460

ctgtaatccc agcactttag ggggttgagg tgggtggatt gcttgagctc aggagtttta 8520

gacctgcctg ggcaacatgg taagacctca tctctacaaa aaaaaaaaaa aaaaaaatac 8580

aaaaaattag ccggtgtagt ggcacgcgcc tgtagtccca gctacttggg aggttgaggt 8640

gggaagatca cctgagccca ggaggttgat gctgccgtga gccataatca tgtcactgca 8700

ttccagcttg ggccacagag tgagaccctg tctcaaaaat aataataata atctggtctt 8760

gagaaaaaat agtatttttt tcttcataaa atattttcca ttttgagaac ttgattaaga 8820

aactcattgt cttgccaatg acattacatt caatcatgct gaaacatcca gaaatagttt 8880

acacatcagt ttgacatcag tattatgcaa tttgaagcca ctgtttgaaa ataaaaacac 8940

tgtaccgtga tttgtttatc cagagttcag attattatat ccttgtatat gagacagaaa 9000

cccccttgta ttctagtgca aactctcttt ggatcttaat atgtatagtt aacaataata 9060

ccatactaca ttctaactac ctagaaagct agcatacctt aacctgatta acttttacca 9120

agttacttga aattatagca aagttaccat ttaaatcttg attctggcca ggtgcagtgg 9180

atgaaccaag catggtggtg cctgtaatcc cagcactttg ggaggctgag gcgggtggat 9240

cacgaggtca ggagattgag accattctgg ttaacacagt gaaaccatct ctactaaaaa 9300

atacacacaa aaaaaattag ccgggcgtgg tggcaggcac ctgtagtccc agctactcag 9360

gaggctgagg caggagaatg ccgtggacct gggaggcgga cttgcagtga gccaagatca 9420

cgccactgca cttcagcctg ggtgacagag cgagactctg tctcaaaaaa aaaaaaaaga 9480

tttgattcta tcagtctact cacctttata gcttgacaat gattgatttg tgtaaaagga 9540

ttcaaatcaa aatttgcaaa ctcccttcct ccaaaggtac tcattttata atactgaaat 9600

tctctattat gttctctgcc cagtgtccca gggtttattg gtttctaaag aggtagtggg 9660

tatatacagc ctccccaagg ggaatttagg aagtaagctg gttgtcacaa agactggcat 9720

taaataggta gagacctagg atgctaatat cttgcaatgt gccaaaataa ttgtccctgt 9780

ccccaacctc accattgcca atattacccc tacccctcac agtgagcgtc acaggcaggc 9840

aacaaactgg tgtcgtcaca gaatgattga tggaacacat agactgcatt cattacctaa 9900

acattgtcgt cacactgcag caaccaaaga caatcgcatt acccaggggt tagatgtagg 9960

aagagtaaaa aacaaaaaat ttttgaatgc gtaattatca ctaattattt tatttgatcc 10020

ttcaggagaa tgtggaagat ggccgagcaa gtatctatca atctgtcgtt accaacacca 10080

gcaaagaaat gtcctgtttc agtgactttc caatgcctga agattttcca aacttcctgc 10140

ataattctaa acttctggaa tatttcagga tttttgctaa aaaatttgat ctgctaaaat 10200

atattcagtt ccaggtattg tatttttggg gaaatgggtt tctctgcatt agttcagctc 10260

atatttagat agaaaagtta ctctgataat gaaagcaatt atgaatgaag tatcccattc 10320

taagtatttg ttgaaatata acagcctcat ataaaaccca aaaagtagtg tcattaccct 10380

tggtattata gattatatac attaattgaa gaggaaaatc atctgttaaa attaaaggtt 10440

tgaataataa tatattgatg tcaaaacttt tttttttttt tttctccctg agacagagtc 10500

tcactctgtt gctcaggctg gagtgcagtg gcatgatctc agctcactgc aacctctgcc 10560

ttccaggctc aagtgattct cctgcctcgg cctccagagt agctgggatt acaggcacac 10620

atcaccatgc ctggttcatt tttgtatttt tggtagggac gaggtttcac catttgggcc 10680

aggctggtct cgaactcctg acctcaagtg atccacccgc ctcggccccc caaagtgctg 10740

ggattacagg tgtgaaccac cacacccagc ctcaaaaatt catttaaact aatatctgtt 10800

atcattgaat acacctagct tcatttgcct tgaaagggcg tataccaaaa ttaaattgct 10860

gttttgtttt cttagcttct tcatagaaat gggatttctt agatgtgtat taaataaatt 10920

cattggtctc tgttcatact agaaggctgt gggaagtatt tgcttatcat ttttttctga 10980

atgcaatctc ttacaaccta aagatggcca gatcattttg aaaaacactt ggaattacct 11040

tttcctgtgc ttcctcaaaa tcaacaaaaa gcaatatttt aattaagcat gctgaatttt 11100

tatcaatggt ctatactttg agaaatagct actatgctta gaaaataaaa tataaatcac 11160

atttcttggc caggtatggt gattcatgtt tgtaatccca gcactttggg aggctgaggc 11220

aggaagatca cttgaaccca agagtctgag accaacctgg gcaatacagt gaaaatctgt 11280

ctctacaaaa aatttttaaa agattatcca ggcatgttga tacccacctg tggtcccagc 11340

tattctagac tgagaaggga ggatcgcttg agcctgggag gtcaaagctg caataagtgg 11400

tgattgtgcc actgcactcc agcctgggca acagtgtgag accctgtctc aaagtaaata 11460

actaacattt ctggataaat aactgttagt gaggcttatt tttaatacat gtcattttct 11520

tagtaattct aatactaggc ttatataata tcaacttaca atagtaaatt ttggtgaaaa 11580

tttgtattta taaattccat taaaatgtcc agttctacct aatgtagttt ttcaccaatt 11640

cctggtagat ctaacttgtg aataacagat tatgtatacc agaaggtttt gtaactttgt 11700

gcacttaact atcaatctac ttaacaaata tattgccttt ttatgatata taacttctat 11760

tccattcttt taaagatcat gttagagtcg caaggaagtc atttctcttg gttattgtgt 11820

tactgctact tttgtttctt ggagagtgaa gaggggttgg gaagaaaggt ttctgtttat 11880

tggtctctga gttggtgtaa gtcataggtg ttagagctca actcgagaag caggcaaact 11940

gtaacaagcc ctgttgctta tgattgtcaa tgtaatctac atcagtgctt ctcaaacttt 12000

aatgtggaca tgaatcacct ggatatcttg ttaaaaatgt aggttctaat ttaataggta 12060

tggggtaagt tctgaaattc tgcatttctg acaagcttcc aagtgatact gaagatcctg 12120

atcctcaaat cacattttga atagcaagga tctacagcac ttagttaata tactactttg 12180

aactaccatc tgaaatcttt tctttcatct gaaaactgcc cagatattta aagccctttt 12240

acaagatttc tactaatatt ccatatacat ttttaaattg agacagctta aaaattacca 12300

acccagcagt tggaaaaata tctgaaaatt tgagatatat aaaagactaa aatacttgca 12360

aatgagaagc atgccattcc tctagcatta taaactttgc ttccacttga catcgtttct 12420

taatccagca gatatgaaac atttatgtac aattttaaaa attaacagac ctccagtgag 12480

ctacatttaa aaaaatcaat gaaccaataa atcattttat tcaaataaga tcatgaactg 12540

tcttgctcac atgatgtact ctgttttaaa aatagcaaat gttaaaaact atcattcagt 12600

ggaatgctga ccatgtgtca ggcactctgc aaagtgtttt gcgtgaaata tcttctctaa 12660

tacaaagtcc acaaagaggc ggctacataa aacgttcctg acatatgcca attgcatgat 12720

cacttgaatt attggtttgt ttccttgttc agattatcaa ataacaaaca gagagaagtt 12780

ctttaaaaga aaagatatat atttggtgat agagcattgt aatgagaatg tacatgccat 12840

ggtaaactat ttgtgtattc agggagttaa aggaagacaa aggtttttaa atggggaaaa 12900

aatacaatta cataattgtt ttgaaataat tatataaaga gcaataacaa gggtgatgcc 12960

agtctgagat tggacagtta ctgagcagat gttcttgtag aagtcatttt tgtgtaagat 13020

tatgatggtc tttgtgtaag gtggtggttt ttgtagtttt tgttatcagg cacacatcat 13080

gagaacccgc tctttctggc ctttcccaat tctatttgtc gggtttctta acattagtga 13140

ctccatctag attctgacag ttttcatgag aacttgcttt tcttttctct ctcaagtcct 13200

tattcagtat tcagcaccct taacagatta gtcccactgc tgagtcaggc ctcttgcatg 13260

aagcagcaat gagaaagaca cacttggcca atgttatcct ggagtaattc tcaatgatgc 13320

cttctctgtg tttcttcaag acaactgtcc ttagtgtgag aaaatgtcca gatttctcat 13380

cctctggcca atggaaggtt gtcactcaga gcaacggcaa ggagcagagt gctgtctttg 13440

acgcagttat ggtttgcagt ggccaccaca ttctacctca tatcccactg aagtcatttc 13500

caggtgagac ccgctgggat tcccagcttt ttggagtagg tttccaggta ctttatatgt 13560

agtttggatt gacaagcagg attcattgct gcaactgggc agaacttggc tcaataagat 13620

tgagacagag ctagaaagat gaaagacacc aaacatcatc tttgtttcta ttggcctctg 13680

agtcttcatc acacatagat ctcagagcca acttccttgg aagtcactaa gtccttggca 13740

taattttaga gaattcacat caaactggtt ctctgttgga gaggcccttt tagccatgtg 13800

cctgcgttgg cctttttcta ccctgccaaa caccgagcct ttttcacagg gccatactca 13860

cacacaaggg gagagctcct agaaagaaat gctttgcaag ttagtgatgg ggagagaagt 13920

gcaggaatag aaccctgcat ccagctgttc tggtccaccc aagtctttcc tcagagaaca 13980

cacttctttc ccaaggccct taggaaaata tgtaatatag tggttcatag tccaggcctc 14040

atattagaat cacctgggga gcttctaaag ccctgatggc ctggagacct acccccaaag 14100

attcaaacac tatggagtag ggttagagca atgaaagttt gctcaggtga ttttaatata 14160

cagtcaggat taaggcctgc tcatctaaag caattgttct caaatagagt cacctggagg 14220

gcttttgaaa gcacaaattg ctaggcccca ccctccatat ttctgattca ataggtgcta 14280

tggcttgaat gtcctgtcca aaactcatat tgagattaat ccccaatggg gcagtatgaa 14340

gaggtggggc ctttaagagg tgattgagta gtaagagctc tgccctcaag aatggattaa 14400

gccatttgtg gataaatagg ttaatggatt attgggttac acaggagtgg aactggtggc 14460

tttataagaa gaggaagaga gacctgagct agcatgttag catgcttggc tccctcacca 14520

tacaatgccc tatgctgcct tgggactctt cagagtccaa accagcaaga aggctttcag 14580

cagacgcagc ccttcaacct tgacttctca gcctccacaa ttgtgtgcca gaagaaataa 14640

cttccttccc ctataaaata ttcggtttca gatattttgt taaaaacaat agaagacaaa 14700

ttaagacagt agctctggca tgaggctgag aatttgcatt tctaacacca ggcaatgctg 14760

atattgctgg ccatgtgacc acactttgag aaccaataat ctaaagattc tttcaagcaa 14820

ccccaccatc aatggcaaat actttataaa gtcatgtgtt tccgtgaagt gtaaaagtag 14880

taactaggaa aggacacaga agaagcttgt ctgtgattaa ccaccagcaa gtcactgatt 14940

tacacaatat ggaaaccaac tcctatgtgc ctggttttta gttttagttt ttgtttactt 15000

tttgaaaata agattgctaa attgtattct aactattaca caattataat aatagcactt 15060

cataatgtgc ttaagaaata tttaagagta tctgataagt gatttttttt ttttttgaga 15120

tggagtctca ctgtcaccca gactggagtg cagtggcacg atctcagctc actgcaacct 15180

ccacaacctc catcttccag gttcaagcaa ttctcctgcc tcagcctccc aagtagctgg 15240

gattacaagt gcacgaccac ccctggctaa tttttgtatt tttagtagag agagcttcat 15300

catgttggcc aggctggttt caaattcctg acctcagttg atccgcctgc cttggcctcc 15360

caaagtgctg ggattacagg tgtgagccac cacaccttgc ctaatatgtg atattaaagg 15420

gtcaaatgtc attatatagt ccaaaatagt atataatagg caggcagaag acagtatctg 15480

gtcctgctgt gttcatcacc atttatttgt ctctgataga gacaaactgc agccgtaagc 15540

tgcagcctct gaaataaaaa atcaacccct ttggtcctgt ttttttgttt gttttttgtt 15600

ttgttttggt gttgtgacag tctcactctg tcacccagac tggagtgcag tgactcaatc 15660

aggggtcact gcattcttta cttcccaagc tcaagcaatc ttcccacctc agtcacccga 15720

gtagctggga ccacaggcat gcacaaccat gcccagctaa tttttgtatt ttttgtagat 15780

acagggtttc actatgctgc tcaggctggt ctcaaactcc tgggctcaat caacctgcct 15840

aggcctccca aagcgctggg attacaggcc ccacctggtc tggtacctaa actttcttat 15900

gtgctttact cctatagaga agaggcaaaa caattattaa ctccagaaag gaaaagctgg 15960

caatgcagtt ttattgaaat tagcttgaca tagttgctct ggagctcaca gacttctctc 16020

ttcttccccc tgaaggtatg gagaggttca aaggccaata tttccatagc cgccaataca 16080

agcatccaga tggatctgag ggaaaacgca tcctggtgat tggaatggga aactcgggct 16140

cagatattgc tgttgagctg agtaagaatg ctgctcaggt gtgatgctct ctgcttacca 16200

tgtacctgga ggggaggaag tggggatgcc atactggaga accccagcca tataatcgcg 16260

gctccaatcc tcattaacta gttggttggt agcgcattgt ggcatcatag aaaatctgga 16320

agtcaagaaa ccactttacc tcctagctct gtcactaacc agccatgaat cctagagtga 16380

ttcatttcac ttctctggga gatggctccc tcatttttaa aatgggaact tttgaccaga 16440

tgattttcca tataagaggc ctttcatcaa catggctcac tgcagccttg acctcctggg 16500

ctccaatctt cctgtcatct cagcctcctg agtagctggg actacaggca catgccacac 16560

cacactcagc taattttcat atatttgtag agatgagggt cttgccatgt tgcccagggt 16620

agtctaaaac tcctgaactc aagcaatctg cccgcctcag cctcccaaag tgctgggatt 16680

acaggcatgc acaaccacac ccagccaaga ggccttgttt ctacctggat gtttaatgag 16740

aggttaatct gttcatattc tggagggtgg cttttagaaa tttagtgtgt atttgaatta 16800

tatttgaaat atagataacc ttcagttacc caaatattat gaaaagaaag attaaataga 16860

tagtaggtct ctcaactaaa atcatagata tttaggtgct tcctgaggcc ttctaaccac 16920

tgtcttcttt gcacctgctc aggaatgaca ccagctgagc tgccaaagag tcaaacattc 16980

attacatgat gatgctgctg acagtggtgg tcaggaatag caaaaactaa actccttctg 17040

caaggacaga cctaggcaaa gaagggaaaa tcactaaaca tcctttccca aagtattccc 17100

tctcaagaag gcctgaacca gatgcccaat cactcttacc ctagctcttt cagcctgatg 17160

tctctggcca cccagggctt accatggccc tgtgcacaac caacaaatca tttccatcct 17220

aagtcttaca ctttcaggac tctagatacc cagtggcaaa agttacaagc aaacatgaca 17280

cccgcccagc aggttaatga aggggttata ctgggacctg tcagagtcat ctatcagtca 17340

gttagttagt gccagcccgg gaacagagca ggtcactaac accggaaaga gacttactag 17400

acccaataag tcttcacttt gtgaaaataa acctcttgtc acttatcacc tcagtgtgaa 17460

gaacaagtga ggaggcagga actgtgacag cctggagaag agcagagctg gaaaatgaga 17520

gtaccagctc taggctcttt catgctacga atacccgcaa agccttagga acagagtgta 17580

atggggcagt atgtgaggag ctaatatagc agtcagccaa gtgaagatcc atcctagact 17640

acttcacgtt gtcagaccag tgatttggat ttagatctct tcattccaaa gatatcaaat 17700

cttagatggc aagaaccagt tccttgtatg ggtcttgccc tacaggaaga cttatggtgt 17760

gagattcaat attaagaaac taccttggct ctatttgcat gccttacagc ttcttaaaca 17820

atcttttgca cagagtgcaa aagactttgt ttccatctcc ctctatcagt gtaaatgcca 17880

ctagatgccc cctttttagg aggtacttca ctttgaggtc aatcatcttt aaaacagagc 17940

ctcagtaaat tctggggcta tgcatgtgat acatcaccta cataatagat tcctcctaaa 18000

tataatgtta taatcataca tttccaggat tatactcatt catctgcact aatctcttca 18060

atatttatta gagtaacaac ataaatctat aactatgata aaacctctta cacagagtaa 18120

tatactctca agccttctgt gaaaagacta accagagact ttacaggagc tatacatgct 18180

aggaacggaa ctaggcgcat ctgcaaaact tgaaattaca acctgaactc accaaaattc 18240

tgagtgtgca ctgctctgtt aaaagaaatt caccttcata aggttacagc accctctacc 18300

acaatccaaa agcaccactc aagatcatat gggatggtgc tgcatcattg tattagtcca 18360

ttctcaacgc tgctatgtag acatacccga gactgggtaa ttcataaaga aaagaggttt 18420

aattgactta cagtttggca tggctgggga agcctcagga aactaacaat catgacggaa 18480

tgcacctctt tacgaggctg caggagaaag aatgagagcg actggggaac cccttataaa 18540

accatcagat ctcgtgagaa cttactccct attaggagaa cagcatggca gaaacctccc 18600

ccatgattga attatctcca cctggtcctg cccttgacac gtggggatta ttataattta 18660

aggtgagatg tgggtaggga cacacagcca aaccatatta gtcatttaca tacttctgac 18720

caaaaaccaa atctctggcc tttgacctaa aacatgcgtc tcagagaaag cagcctgagc 18780

ctaaatcctc atgtttctct cactgttgca gctagtgtca ttaaggcagg ttagaccacc 18840

ctgctgtagg gagggtcaca acagaaaaag agtgaatcaa acgggcagag cataccattt 18900

gaaacatggt ttgctcctga gaaagaagag gggacagtaa gtaatggaaa gagacactaa 18960

tgaaaatatt tttgtatcta atatctaatc aaagtattgc caagtcagcc tataagggca 19020

acggcaggag aaattcagaa cataggtata taccacacac agaccagcaa tataggaatg 19080

cttggtatag gtgctacttc acaagctagg aatgtaaggc ccatccccac aaaatttgtc 19140

tccaaattct ggtttactcc agacataagg cactgtatga aactcctctc ttccagccta 19200

actttataac ttaacagcta gcagtactta tcacttgcca ggcaatattt caagtacttt 19260

atatatacca cctcatttaa tctacacaag aatgccatga ggtaggtact gttaataccc 19320

ccattttaca gagagagaaa ctgaggcaca gagagattga aataattcaa ccatggcaac 19380

acagattgaa atagttcacc cacagtagtg tgattgggat tcaaacccaa gcagtctgta 19440

tccaaacctc tcaagtaaat tggttacctt gcaagtgaat cttatgtgtt tatcaagtat 19500

agccttaaac aaaaacttat tgcatggtat gtaaaaattt aagaagcagt tcaagtatgc 19560

atttggccaa tgggggagta acagcaaaca cagcaaaata tacatttgaa aagagattaa 19620

atgtacattt tggaaacaag ggaaatctta ataaacaagg taaagaatac acctgaaaga 19680

ggattcagat gtgcacttga agagaaagag aatcacagta taagttcaga gtttttaact 19740

tttaaaatac attacaagca ctgtgtctca tgcctgtaat cccagcactt tgggaggctg 19800

cggcaggagg attgcttaag cccagaaatt tgagaccgac ctgggcaaca taatgagacc 19860

gtctctacaa aaaaattgtt tgaattagct ggatgtggtg gtacatgtct gatactgagg 19920

tgggaggatc acttgagcct gggaggtcga gactgcaatg agctatgact gcacaactgc 19980

agtccagcct gagtgacaga gcaagaccct gtctcacaca cacacacaca cacacacaca 20040

cacacacaca caaaataaag tcttttaagt atggaaggaa gattatttcc cctgttattc 20100

tccatccagg gatattcaga tgcatataca cttatacttg tgtagtcact aggctataat 20160

cgcacatttc caaggattat aatcattcta cctgcactat agaagaaact taggtgagtg 20220

gaaaacatga gaggagggag ggaggaactt tctcttaagg agcagcaaac cacaactgta 20280

aacatgggaa agacttgtgg attttatcat cagagttagc ccaaagactt tctcgtgtct 20340

ccatgaagtt ctcaagattt tgttgcagtc ttcctgcatc agtgtaaatg ccactgggta 20400

cccctattta ggaggtactt tacattgagg tcaatcatct ttaaaacaga acctctgtaa 20460

attctggggc tacacatgtg atacatgacc ttcatagtag attcctccta aacgggacaa 20520

tgccctaatt taaactgcat ttctttttgc ttgccaggtt tttatcagca ccaggcatgg 20580

cacctgggtc atgagccgta tctctgaaga tggctatcct tgggactcag tgttccacac 20640

ccggtttcgt tctatgctcc gcaatgtact gccacgaaca gctgtaaaat ggatgataga 20700

acaacagatg aatcggtggt tcaaccatga aaattatggc cttgagcctc aaaacaagta 20760

gagttatttt gcttttttaa tggtatactc gttggtgagc aaagttgtct gaaggtgtct 20820

cccttaacaa agattcaaat tgctaacacg gtagttaaaa ctacaatcta acaatatgag 20880

tatcttatag gtcctggagt ttagcttcta aatttggtct gtatgccttt aaaaaatact 20940

taagaagatg aagcagaagt gttataagct gctccagaaa gcaaaactag gggagaactt 21000

tctaataccc agagttatct aacattggag aaaactgttt caagagatta cgacctgcct 21060

ttcagagggg tgtggtggga aacatgtaat tctccatcta ataatttatg ctttgctaac 21120

cctatagcat gaaggttctt cccatgggaa acctttgaaa acacattcct ttttctttgc 21180

taaaagacaa atctctgttg acgtcaaagt tatatgtcag tgatttaagc acaagcaaat 21240

gttatgaatg gttcttttgc tttagttgtt acaggcttct tcccttaaaa aaacagaaga 21300

gctttagaat cttttaacaa atgcctgccg tgcaactacc atattctaag atctgacata 21360

agtgccacgt atcgtctatt aaaaaaagaa aaagaaaatg ttctcaaatc tacaaaaaaa 21420

ataagcggac tttgcatcaa catccatgct attactaaca gagactccat ggatatttgg 21480

gattaacaaa tatcaccaaa cctaatttta tacattaatt ttcacattga tcccttcata 21540

gatttcaaaa ctagtggaaa tttagcaaat tttttcttat gatcaaatag gggttaaata 21600

aaacagcaaa ataataaaag ctagatagca tgaaaaaggt taaaaacaga aatggtataa 21660

taaccaccat aatacttggg gattgaccat aggcacaggc attttgtcta agcccttggg 21720

gatgcttcct tccttaaaat ctctttcact cacgttgcct acatgttttc ccttatttat 21780

tgacaagaga tatttgtgac atgagaatta agtcagaaaa taaggatttg cacagacaac 21840

cagttaagtt agagttttac agatatttga aaagcccttt tattttcaga gccgtacccc 21900

aaaaatatca agagggttca agattcctca gcaaatgatc cttcagaatg tttttcttct 21960

gtatgtctca gatacattat gaaggaacct gtactaaatg atgatgtccc aagtcgtcta 22020

ctctgtggag ccatcaaggt gaaatctaca gtgaaagagc tcacagaaac ttctgccatc 22080

tttgaggatg gaacagtgga ggagaacatt gatgtcatca tttttgcaac aggatatagt 22140

ttctcttttc ccttccttga agattcactc gttaaagtag agaataatat ggtctcactg 22200

tataaataca tattccccgc tcacctggac aagtcaaccc tcgcgtgcat tggtctcatc 22260

cagcccctag gttccatttt cccaactgct gaacttcaag ctcgttgggt gacaagagtt 22320

ttcaaaggta agtgtgtagg caggtgagtg gctaagcgtt tcagatctgg tgaagtttat 22380

caataatgat aagaaggttg cctgagataa aaaggttgcc aagaaaaagt ttgacaacct 22440

tggctgctct cacaagacta acattctaaa aagttactgg agaattcaaa gaataacaaa 22500

tacaggaatt tagtaataat aaatacctgc aatcatcctt ttaaaatatt agacagtcaa 22560

gagaatttca actggcataa agctaagtgc atgttaactt ttctttgaat cgtgagagat 22620

aagtttaaga aaaagatctg tctcctggtt ttacctctgt gttgtttaaa aattcctcag 22680

catatctgca aatcaattta actcttaata cttgagcagc tcaacctcac aaatccctac 22740

aagttataaa attattaaaa ggtttctttc tgggtgtctg tgtagcactt catactcctc 22800

agaacggtgt tacctccctg cctccagggt tcaattctgt tcagcaaaag cttactgaat 22860

accttgccct gtgctgggaa ctggtgggac agagagaaat ttaaacagat catttcaaca 22920

taacatgaca aatgctttga ttgaataata tatggagtgt tcagggaagg agagaaaggg 22980

cacttatcat ggtagaataa gggaagggca catgataaag gaaaacgtcc tggataactg 23040

catttctcag gggcagaaaa ggggattgcc tgaacaaaag catagagtca atgatgcata 23100

tggaagggca catgctattt gacattgcta gagcatgacg tatgaggcag agagagatga 23160

gccattactc ttggagaaga aggagacagg acacaggaat tttttaagac atgctatgga 23220

gcttagatta taaattatag atcagttctt cccaaatatg gctacatatg aaaatcatct 23280

gatggatcct tagggaccct gattaagtaa gactggccaa gggacctgga atctgcattt 23340

tagaaagctc ttcagcccgg ggcaccaatg aagggttata agcaaggaac aggcattagc 23400

agatttacac ttcagataga ttgtttcagc agtagtgtgg aatatagatt tgaaagtggg 23460

gaaagactac agcctcaggg atgaaagaga aagctactga aatagcctat gctaaaatat 23520

gatgcatcct gggccagggc agagatacaa agtggaaagg aagccataaa tgtgagaaat 23580

cattaaggga aaaatcagca tgacattatg attggttcaa tgtgggaaag tcagagaaat 23640

agagaggaat ctaggaggac ttacagatct ctggcattgg aaaccaggtg gacagtagtg 23700

ctgtgaatac agagggggtg tgcagaaaat gatgcaagtc tggacaggag ggcttcagtg 23760

aggagctcag gtctggacta cttgaacatg agatgtctga tgactctagg caaggggact 23820

tgaccatatt tcaacacatc caaagctcag gggacacttg tgggcaggcg atggagtcat 23880

gagcacacag taataacttc tgcatcaatc tttccctatc tctactgccc tactctcatc 23940

tctcaccagg tttatttcaa cagcctcttt actggtctcc ccagctttgg gcttgcctcc 24000

ctggagtcca ttttcctaaa ttcagcagcc agacagatct ttccaaaaaa taaatctgat 24060

cttctcactt cattcagaat actcttccac tgatttgatt tggggcctcc tgtcaccttc 24120

aggatagagc ccaaaccact agtcatggct gccaggctcc cagacacact tcccttttcc 24180

agcctcttct cttggccctc tccacttgta gtccatgccg tagactgtgc accctggaca 24240

gtgtcacata gagtgctatg ggggtggcac cccctgaagt tcaacagcac ggaagccctg 24300

actggtatga catggttcaa tgtccagagt ttaattttaa gaatcaacaa ctagacaaag 24360

taatgatatt gactcaaact tactattcaa accaaccttt tattccttag gcttgtgtag 24420

cctgccctca gagagaacta tgatgatgga cattatcaaa aggaatgaaa aaagaattga 24480

cctgtaagaa ttttttttaa ttctttacat gaagcagtgt ttctcaaagt acagtgatct 24540

aactacttac aagaaccacc tagctgcctg ataaaatgca aatttctggg ctatagccca 24600

gatgattgaa tcagaaactc cgtgtgtgag gctaaaaagt tgcattttta tcttcttcct 24660

aagcgattct tatacatact aggttaagaa ccaaatactt aaagataaga attgtaccaa 24720

atcagagcac ttctccttgg cttaatttca tttcagtttt atatgatgcc tatgtcagat 24780

tccataactt ctcaagccac ctacactctg tggttagaga gggaatggga tgagacagtg 24840

gtggtgatag tagcttgaat agctgtgaaa agttagagaa tccccatcag aataaattag 24900

gaaggggttg gtgtgaaggt tcaaggattt gtactttgtg atgaggtaaa atgaggttca 24960

acagtgatcg agtacccttg gaaagttgat ttggggctta catcaggtgt aaagagtttt 25020

ctcatgttca aattcaaatt tacctaagat tgattgagta tctactatac gccatccaga 25080

ctgccaggta ctttagtaat ttaacaagca aatattaagc atctcctttg agcaagacac 25140

caagctatgc tttcatatgc attatctcat gaattcctgc agccgccctg gctagcatgt 25200

acttgcctgg agatttgcca ccgcttaaaa aatgccaaac aatggttacc aatcttgtca 25260

catttctaga gcatccatga attcatggct ctttatttga gggcgtattc tcaatctgag 25320

atatgagcct cctggtatga taaactcaaa ctttccacca gagattcatt gaaaactcat 25380

tcacatattc actcattcct tcattccttt agcagttttg aatgcctaat attctagaaa 25440

acttagaaca ttctgtgaac attccctttt tactttcttc actaaggttt ggagaaagcc 25500

agagccagac gttgcagacc aattatgttg actacttgga cgagctcgcc ttagagatag 25560

gtgcgaagcc agatttctgc tctctcttgt tcaaagatcc taaactggct gtgagactct 25620

atttcggacc ctgcaactcc tattagtatc gcctggttgg gcctgggcaa tgggaaggag 25680

ccagaaatgc catcttcacc cagaagcaaa gaatactgaa gccactcaag actcgggccc 25740

tgaaggattc atctaatttc tcagtttctt ttctgttgaa aatcctgggc cttcttgctg 25800

ttgttgtggc ctttttttgc caacttcaat ggtcctagtc agcataatgc tttgggcttt 25860

attatcttgt cagtcactac ctcctaaaga aaaaaaaaaa ggctagaaga aaaaacatta 25920

cattcatgtt ctaattatag attttagagt taggtagtac aggtaagggg gaaattgtaa 25980

agaattagca gaattaggca tatgtacaaa accaaa 26016

2

aaccaaggga gaaaactatt ctgtcaaaga gacggtgcca aaaggcaaaa acaaaggagc 60

tgatggcaaa gaaggtagct gtgattggag ctggggtcag tggcctaatt tctctgaagt 120

gctgtgtgga tgagggactt gagcccactt gctttgagag aactgaagat attggaggag 180

tgtggaggtt caaagagaat gtggaagatg gccgagcaag tatctatcaa tctgtcgtta 240

ccaacaccag caaagaaatg tcctgtttca gtgactttcc aatgcctgaa gattttccaa 300

acttcctgca taattctaaa cttctggaat atttcaggat ttttgctaaa aaatttgatc 360

tgctaaaata tattcagttc cagacaactg tccttagtgt gagaaaatgt ccagatttct 420

catcctctgg ccaatggaag gttgtcactc agagcaacgg caaggagcag agtgctgtct 480

ttgacgcagt tatggtttgc agtggccacc acattctacc tcatatccca ctgaagtcat 540

ttccaggtat ggagaggttc aaaggccaat atttccatag ccgccaatac aagcatccag 600

atggatctga gggaaaacgc atcctggtga ttggaatggg aaactcgggc tcagatattg 660

ctgttgagct gagtaagaat gctgctcagg tttttatcag caccaggcat ggcacctggg 720

tcatgagccg tatctctgaa gatggctatc cttgggactc agtgttccac acccggtttc 780

gttctatgct ccgcaatgta ctgccacgaa cagctgtaaa atggatgata gaacaacaga 840

tgaatcggtg gttcaaccat gaaaattatg gccttgagcc tcaaaacaaa tacattatga 900

aggaacctgt actaaatgat gatgtcccaa gtcgtctact ctgtggagcc atcaaggtga 960

aatctacagt gaaagagctc acagaaactt ctgccatctt tgaggatgga acagtggagg 1020

agaacattga tgtcatcatt tttgcaacag gatatagttt ctcttttccc ttccttgaag 1080

attcactcgt taaagtagag aataatatgg tctcactgta taaatacata ttccccgctc 1140

acctggacaa gtcaaccctc gcgtgcattg gtctcatcca gcccctaggt tccattttcc 1200

caactgctga acttcaagct cgttgggtga caagagtttt caaaggcttg tgtagcctgc 1260

cctcagagag aactatgatg atggacatta tcaaaaggaa tgaaaaaaga attgacctgt 1320

ttggagaaag ccagagccag acgttgcaga ccaattatgt tgactacttg gacgagctcg 1380

ccttagagat aggtgcgaag ccagatttct gctctctctt gttcaaagat cctaaactgg 1440

ctgtgagact ctatttcgga ccctgcaact cctatnagta tcgcctggtt gggcctgggc 1500

aatgggaagg agccagaaat gccatcttca cccagaagca aagaatactg aagccactca 1560

agactcgggc cctgaaggat tcatctaatt tctcagtttc ttttctgttg aaaatcctgg 1620

gccttcttgc tgttgttgtg gccttttttt gccaacttca atggtcctag tcagcataat 1680

gctttgggct ttattatctt gtcagtcact acctcctaaa gaaaaaaaaa a 1731

3

Met Ala Lys Lys Val Ala Val Ile Gly Ala Gly Val Ser Gly Leu Ile

1 5 10 15

Ser Leu Lys Cys Cys Val Asp Glu Gly Leu Glu Pro Thr Cys Phe Glu

20 25 30

Arg Thr Glu Asp Ile Gly Gly Val Trp Arg Phe Lys Glu Asn Val Glu

35 40 45

Asp Gly Arg Ala Ser Ile Tyr Gln Ser Val Val Thr Asn Thr Ser Lys

50 55 60

Glu Met Ser Cys Phe Ser Asp Phe Pro Met Pro Glu Asp Phe Pro Asn

65 70 75 80

Phe Leu His Asn Ser Lys Leu Leu Glu Tyr Phe Arg Ile Phe Ala Lys

85 90 95

Lys Phe Asp Leu Leu Lys Tyr Ile Gln Phe Gln Thr Thr Val Leu Ser

100 105 110

Val Arg Lys Cys Pro Asp Phe Ser Ser Ser Gly Gln Trp Lys Val Val

115 120 125

Thr Gln Ser Asn Gly Lys Glu Gln Ser Ala Val Phe Asp Ala Val Met

130 135 140

Val Cys Ser Gly His His Ile Leu Pro His Ile Pro Leu Lys Ser Phe

145 150 155 160

Pro Gly Met Glu Arg Phe Lys Gly Gln Tyr Phe His Ser Arg Gln Tyr

165 170 175

Lys His Pro Asp Gly Ser Glu Gly Lys Arg Ile Leu Val Ile Gly Met

180 185 190

Gly Asn Ser Gly Ser Asp Ile Ala Val Glu Leu Ser Lys Asn Ala Ala

195 200 205

Gln Val Phe Ile Ser Thr Arg His Gly Thr Trp Val Met Ser Arg Ile

210 215 220

Ser Glu Asp Gly Tyr Pro Trp Asp Ser Val Phe His Thr Arg Phe Arg

225 230 235 240

Ser Met Leu Arg Asn Val Leu Pro Arg Thr Ala Val Lys Trp Met Ile

245 250 255

Glu Gln Gln Met Asn Arg Trp Phe Asn His Glu Asn Tyr Gly Leu Glu

260 265 270

Pro Gln Asn Lys Tyr Ile Met Lys Glu Pro Val Leu Asn Asp Asp Val

275 280 285

Pro Ser Arg Leu Leu Cys Gly Ala Ile Lys Val Lys Ser Thr Val Lys

290 295 300

Glu Leu Thr Glu Thr Ser Ala Ile Phe Glu Asp Gly Thr Val Glu Glu

305 310 315 320

Asn Ile Asp Val Ile Ile Phe Ala Thr Gly Tyr Ser Phe Ser Phe Pro

325 330 335

Phe Leu Glu Asp Ser Leu Val Lys Val Glu Asn Asn Met Val Ser Leu

340 345 350

Tyr Lys Tyr Ile Phe Pro Ala His Leu Asp Lys Ser Thr Leu Ala Cys

355 360 365

Ile Gly Leu Ile Gln Pro Leu Gly Ser Ile Phe Pro Thr Ala Glu Leu

370 375 380

Gln Ala Arg Trp Val Thr Arg Val Phe Lys Gly Leu Cys Ser Leu Pro

385 390 395 400

Ser Glu Arg Thr Met Met Met Asp Ile Ile Lys Arg Asn Glu Lys Arg

405 410 415

Ile Asp Leu Phe Gly Glu Ser Gln Ser Gln Thr Leu Gln Thr Asn Tyr

420 425 430

Val Asp Tyr Leu Asp Glu Leu Ala Leu Glu Ile Gly Ala Lys Pro Asp

435 440 445

Phe Cys Ser Leu Leu Phe Lys Asp Pro Lys Leu Ala Val Arg Leu Tyr

450 455 460

Phe Gly Pro Cys Asn Ser Tyr Xaa Tyr Arg Leu Val Gly Pro Gly Gln

465 470 475 480

Trp Glu Gly Phe Arg Asn Ala Ile Phe Thr Gln Lys Gln Arg Ile Leu

485 490 495

Lys Pro Leu Lys Thr Arg Ala Leu Lys Asp Ser Ser Asn Phe Ser Val

500 505 510

Ser Phe Leu Leu Lys Ile Leu Gly Leu Leu Ala Val Val Val Ala Phe

515 520 525

Phe Cys Gln Leu Gln Trp Ser

530 535

4

25464

Homo sapiens

genomic

4

tccagtcctg cagcaacctc ctagttcctg ctctttcagc tctttgacct tttgcaagca 60

cctaattccc tgtagtatat acctttcttc atgatatata gtgtttttta tctcctgcac 120

taaatcatga gcatatgcat ataaatcata atatgaaatc ttaaaaacag aagtactttt 180

gctgaggcat taagcatata atcagtcagc aggtccccaa acatctaatt cctgaatatc 240

tcatatatcc tgtctccatt atccattcct ctaatgctac tctaatttaa gtcctcagtc 300

tctctggcct agattgttga aataacatcc tgggtttttg gtctccttga ttctagtcac 360

catcctctct agcctccagg tgaatctgat cttgtctgat gttgtcactt ccttgttcaa 420

aattctcgaa tggacaaccg taatccagaa ggtagtatcc aaacctgtga ttgtggcact 480

tcagtatcct tcataaccta tgtcctgcat gtttaaccca tattttgcta ttcccatcac 540

ttatggtcca gcaaaactga actaattgta gttcccccat cacgtgttct tacttttcta 600

tgcattttca catatttttc tctctgcctt ttttctattt cttgtccctt atctgtctgg 660

aaaacatcta ttcttccttc aagactcagc tgtcttctca cactccttga agcctctctt 720

tcctcctcca agtggaccta gatttttctt cctacatgct agcactacac tgaaccatac 780

ttccactgtg acatttatca tctccctcaa cactagactt catggttcca gatggaaagc 840

actgtgtctt ctcacctttg aatcccccaa aagactatta taatgcatga catatagtag 900

gctgtcagta cagtgaaagg aatggccaga ggaaggaaag gagggaaaca gaagcagaaa 960

ggacaggtat agaagccgga gggagccaga gacaaggttc agagaccaca attctgtctt 1020

ttgagttcac tagttttaca agctcatcta taagcgttag ttcagcaact cagatcaggc 1080

cctaagtttc cagaaatttg agctactttt cactgttggc acaacaaaac gtttcattat 1140

agtccaggtg catagccttt gtttatatat tctatatttc caaagcaaac ataaatgaaa 1200

gaatcattgt tcccctaatc tcccaggagt ttcaccttac agctccagtg gccatggcag 1260

tcactgtttt atattttttg taacaagaac caaagacttc attcttcctt tttcctaccc 1320

ctttcttttt acttcaccca tgcctcccct gttcttctct tatccctacc acactcgtcc 1380

ttctctttca gattttacta tggctctata ccattaaaaa tacaagaaaa aaaaggaatt 1440

ttactttaag aataactcct cccccttccc cagttttcac atcaaaagac attgttaaat 1500

gccattctct tccacatttc gagaactgct gattctctgg ggagagaaag gtgattgctt 1560

aagaggtgaa gtcccttaga gcattcaaaa tgaggagtga ttctgtacag aggatatcat 1620

gcagcaggct ggatgtctag ttccaattcc tttatttgtt acctctgaga ccttgaagaa 1680

gtagtttcta gtctcagcat accaaagcgt catctgcaat tgagagcatt ggattgatga 1740

tcttcaaggt ccttcctgct ctagcattca ctgaatctgc tatttttgac atattgaata 1800

atcagaagca gccagtttta gaatcttatt atagcaaaag tggtaaaaat aatgagcata 1860

tactatcaat gtgcatctat gtcttcttat gtttgagtga ggatcctgat acataaacct 1920

tggctgataa tttctactga aaaaaatcgt aagtattaaa gacactcttc tgaagatgtt 1980

ctctccagac tctgctacag gcaatcatga gcaagagggt tggcatcatc ggagctggag 2040

tcagtggctt ggctgccata tggtgctgtc tggaggaggg gctggagccc acttgctttg 2100

aaaggagcga tgatgttgga ggcctgtgga aattctcagt gagtggcaca tcattagaac 2160

accagtggaa ggagatggat tccaatgcaa atcaaatctg atcagttcta attcagattt 2220

agaaggcaga tcacaaaagc tccaaatctg gaaagtaaaa tcttacctct ccaatcatac 2280

taatgcccaa aaaaactatt tcataccagc aaaatttgtc ctgaaaagga cattttcagc 2340

tcattaaaca tcatcacctg catggtgaaa tccagatctc caagctgtaa agggcactaa 2400

tgttggtaat tagtcaaaaa tataccatgg gcttcccagg taagtgaaac aattctattc 2460

tttattgctc ttaaatgcca ggaacacgac tagaaaagag acaaacaaac ctggactgag 2520

atcctgaggt cagaagtcct gagttctaat ttcaacttgt aggttttcta ggcagataag 2580

atttcagtcc agttgctttt gtttccctgg acctcaaatg ctcatttgtc aaatgcagag 2640

gatatgattc tataattaac ttatgtctat tgggcagata gaaattatta tagatgatga 2700

ttgtgtgtgc ggctgttgaa tagcctatca gctccaaatc cagagggaaa aattatggtc 2760

tttgccattt gggctcattg tagaaataat ataattagga aatagtcctt gtaaacacat 2820

ttttttttaa atttcaaagc caagtttgga gaaacttcta gttcttctgt cctggatttc 2880

ccagccattg taatcagttg tcgatgatac atatttggct tgaaaacata ttcacatcat 2940

tcatattgta actacttcct gtcctggtct cagttactgc tctgcctgcg ccaatagcct 3000

cctccaatag agtatatcag tgctaactta gaacacattt ttattcttct ccaagctttt 3060

tttaaaaaaa attgtggttt tgtaaccctg aaagcactcc atgagatata aggtcattaa 3120

tttttatttc ccagtagggg gtaatcaaga gttaatattt ttcaagaatt taattttccc 3180

tatttacatt tgctcaggga aatgtggaca gcttagagta aatcataaaa tggctttcta 3240

ccatctccct agtaacaatt aaatgatgct tgagcatcta ttctggtagt ttgtgctaag 3300

tactgggatg acaaatatgg aatataatca ctccttgtaa atggttccat ttcatttgat 3360

taagcaagcc ataatataat tccgtaatcc tttgatagca aatgggcaaa aactcatttg 3420

atagccgaac ctcttctgaa atcgtaaggt taaataccgt gaattggatc aacatgaagc 3480

taagtctcac cttctgttgc acggcagaaa ttttattgca tttgacagat tgctgcccca 3540

gatctcacta ggagtattat ggagcaaaat ccaaaaatgt acacattcca aaatatatct 3600

ggccctaaga cttttaaaat aagagattat ataactacaa caacaagata gaccttgtca 3660

ccatcaattc aatggacgag tgcctcgagc gtttagaaga gggtggacta cggaaatctt 3720

agtaggtcaa agaaaacctc ccacagggat gacacttagc cttgaaggat aaccccagac 3780

aagcaaaata gaggaccacc tgtgacacaa ctcctagaga gtgcatttcc caataaagtc 3840

tgcgaatggc actccatagg cctatgcagt cattggcagt gtgccagcac caggttaaga 3900

gagaccaaca atccatgaaa ggcacagaaa aggcaatgaa catggtgtgt gcagagaggg 3960

atccatgagt tatccaatat agccagatca gaaagtttac ttaaggaagc aataatatga 4020

tacaaagatc agtaagattc aaagttggat tctgagttat ccacaagagg aaattcttct 4080

tttccataag gtcatgtcta taagcaaaat tctactcaaa gtcctggtga ggatatggac 4140

ccatacaaat actcaaaact ttagcctcct ccacataccc cagcccttcc ttcttttctt 4200

agaaaagttg cttggcacaa tatataatca gagagggatt ttttttatgt gttacataag 4260

actttatctt gtaagccttt tttagaaggt gttctagcag acagaaacgt ggtaattctg 4320

aacttttcac tatttgcttt ttctgagaaa tgaaaaccaa atgggattta aatactagca 4380

ggctgaatgt gtgttttaag tttcatccac tcctaaatag ggcctcgtgt cctcaaaaga 4440

tttcattact gctgtaataa gaagttgctc aacagccagg tgcggtggct catgcctata 4500

atcccagcac tttgggaggc caaagcgggt ggatcacgag aggtcaggag ttcaagatca 4560

gcctggccaa cacagaaaaa ccccatctct actaaaaaca caaaaattag ccaggtgtgg 4620

tggtgggtgc ctgtaatccc agctactcag gaggctgagg caggagaatc tcttgaaccc 4680

aggaggcaga ggttgcagtg acctcagatc atgccactgc actccagcct gggcaataga 4740

gaaagactcc attaaaaaaa aaaaaaatgc ttaccaatag gttagtagca ttttgattgc 4800

aaaagctgaa gccaggacta tttgaacttt ttcccactca tttattcctt tgttcattca 4860

atgaatacat actgtgtact ttatgtgtag ggtactatat taagcataag ctgcagataa 4920

gaggccagcc agcactttaa aagccgtgag aaaacaagta tcagaataac tataagtgac 4980

tatataatta gggcaataag gataatggga ccttagtaaa actaaagatg atttggcagt 5040

agctgagagg gaaggtaaag aaagccatga caaagttgaa ggcaactttt gagcatattt 5100

caagggcata tttagacaag gagatatggg actcataagc agagctggaa taggaaagaa 5160

gatcaaggta aactgcttag atgcatgtac aacattctga aattaacctc tgactttgcc 5220

ctcaagttac ttatgttctc gtgggaaaga tgagagatga acacggttat catccaagac 5280

agatggtgcc cacagctgct tagatctctg gttccagggt aaagctccct cagctagagg 5340

cagagtcaaa gttgaatttc ctccttactg gctcaaacca cacctcatat tgaaataata 5400

aaaatgcatg ctccctggag caactgactt gttatctaat acatttgctt ttttgtgttc 5460

acttggagaa cagtcttttc ggaaaaattc caaggagctg tagtgtacat actcttctct 5520

cctggtgtta taattggctg aggtcaaggg gcaaaaaagc agagattcat tcaagatgga 5580

aatattccaa ggccttagca tctgtttccc agaacagagt cttacattct ttaaccaggc 5640

tccatcccac agttcagccc tgcctccttt caacaggcag ctgaaaaaac ctccttccca 5700

cctctccttc tcacaaccat cagtagaagg cgctagctgt gggtgaaagg gaagcactca 5760

gcctgccaaa ctgctggaca tgagccttca ccctttttct gacctccaca aaaattttaa 5820

aaagtttaaa ttcctgtgct tccacgctta tgagaaatac agcaaccatg aatagaggaa 5880

gattatgttt tcaacttgag aaaaaatact gaggctttgg gcagcccccc acttccccac 5940

ggggacacaa tcctctcaac cctttccagc actttttgtt tccctcttcc agaggtcatc 6000

tggtgtgaga gggagataca catcttgaat ccagcagcaa cgtgacattc catctctttc 6060

cccccattgc acaagagtcc cttccggacc tcgggaagca gaagctgcca gctctgaaat 6120

gtattttcaa ggcagcacat tgtgtgcact tttaccctac cctcacaact gagaggaaat 6180

gtttattttc aatttagctt ttgactgctt ctaaaaaata agccactttt caattacaca 6240

gaggctttaa aatgaagtgc caagatttaa cacatgttct aagggctctg gtttcctgtg 6300

tttctttggt gaggagtgaa gtccagcaac tggtgagcca aagaatagga ttcatttaca 6360

acagagcagt ggttctcaaa gtgtggttcc taaaccagcc acatcagcat caccaggaac 6420

ttgatagaaa tgcaaaccac cccagactcc accccagaca gattgaatcc gaaattctaa 6480

gaatagggcc caagaatcta cggtctaggg agcttccagg cgattctcat tacgccaaag 6540

ctgggaaacc actgcaatat tgggttgttg ccagtgaaga gtttgctaaa ctccaaaagc 6600

aaataaatag gctagaagtc agagcctctt ctagacagtt ttgttttttg tttttttttt 6660

aacctgagta taagatcaga accagtggtg gcacaggaga aagcaaaaac cactaagtgg 6720

ctataaagac agagctaaca ctgagggtaa ttacagtaag aggattcaca tggaaagagc 6780

tccagttctg tgccaggtta cgcgaagggc tttccattcc ttatcttact gagagctttt 6840

aatttttgtt tacgctttta aacatgaaaa gggttttagt caaccaagaa ttgaaccact 6900

gtgttcactg aagggaacac aattcttggc tttctcttta agctttctta ttctccctag 6960

gaccacacag aagaaggcag agccagcatt taccagtctg tattcacaaa ctcttccaaa 7020

gaaatgatgt gctttccaga cttcccttat ccggatgatt acccaaacta tatacaccac 7080

agcaagctcc aggaatatat aaagacatat gctcaaaaga aggatctttt aagatacata 7140

tagtttgagg taggggtctc ataacttgta ctgttgaaat taagatatgt gtgggttaga 7200

gaaaaaggag gcagcaaact attataaaaa ttagagccaa atgtttgggc acctcagtaa 7260

tcaaatgttg ggtctgatta taaagcattc atgcattgat tttttctctc ctagacttac 7320

tagttcacta gtctctgaga gctttcagac taccttagaa aatggaggca gctagcccat 7380

cattgtccac tttccaccct catgctctga tgttttggaa ataatccaaa atgctttagt 7440

atatattagg aattttgtca gttcaatgcc aatgagttgt ggttcaaaaa accagagcat 7500

ttggtagggt ttctcccatt acattatgaa aaggttaaca acttaaatgg gaaatatagt 7560

cattgccccc atctttaccc actcagttca ttagtttttt tattaaaaag gtgagatttc 7620

agcattgttt ctgcgagaat aatgttttac atttatttgg gactctttat tgagcatttc 7680

tgtctgtatg tttggaactc ttaacctcaa ttaactgctg ctaaatgcag aacacttgca 7740

tatagtggga aaaacaatca gcaaaattat gaaccatggt gatatttaca tcattatttt 7800

acctggagta gccccaaatg tatagttaaa ataaaatttt ccaatagtca ttttattcca 7860

ttcattcatt acattcattt gcttccatta tggtgttaat atcaacaaac attaatgaag 7920

ttcctattgt gtgcttgcat tgtgctatgt gttatatgta aaagaaaaag aggtctaaga 7980

cttagctctc aagaagttat ttcaaaataa atatgtaaag agtaagtaaa aagattccag 8040

taacaatttc aatcaaagag aaaatttttt aaagctcttt atgatttgtt tataaataaa 8100

acaatgctat ggagatcatg aagcaagagg caacactttg ggggaaggta ttttctagag 8160

gaggtaaaat ttagttgtat ttagtaggtg ttttagataa atgagtggca tgagtaaaat 8220

tagagaggtg ggaaaatgcc ctgctcattt ggagaacagt gggcaaacca agttggttag 8280

gagggagata tatatgctag gatgagatat ggccacatat atcagtaaac tagtgtgtac 8340

tgtgactttg aaaaatagag gattattttg caaccatgta aaagaagtcc aaagaaggga 8400

catccagagc ttatgtgatg gcaccaaagt tatcaaagat tcagcttcac ccatcttagc 8460

acgtggccta catcatgacg tttgccttgt ggtgcaaaac agttgctgaa gcttgagccg 8520

tcacatctgc cttctagcaa aaaaaaaaaa aaagtaaaga atgaagggca aagggatgtt 8580

ctctcagctg aatcagctcc ccttttacaa attctcctga aaaaactgtc caacattgct 8640

tatatctcac aggccaccct agttgcacag gaacctggaa aatgcatccc ttttctgtgt 8700

atgttgtcgc tccaaacaaa atcagggttc tgttagtaag aatgaaggga gaatggacat 8760

tagggaagca atttgcagaa tatgttccag aaaagtctgt gggaataaca gaaaataaaa 8820

ctaaaagagt aaattggaac aaaattgtat ggacttaata gtaatcgcat tcaaaatgta 8880

gaataagttt tagaggctgt gaagtaacag aaattgagca gtgaattgag cagagaaatt 8940

gagaaatgaa tatagtcctt caggaagatt aatctgacaa gcaggacaaa ggatggcttg 9000

taggaaatgg gaggctgaag acaggctagg tataggttct tgccgtagtc catgcaaggg 9060

agtgataagg acttgaatga aggcagtgtt agcaatcatg gaaagaaagc gtgagattgg 9120

gagataaata ctgtttaaac atgaggcaag gatggagaaa taacaaggaa aacaagtcat 9180

ggatttgaag cataagtggc tgggagtttc atgtcatcat tcaaagaaat aagaaagtca 9240

gaagccagtt tcaaaggaaa tttaagtagg tcaatcaaaa cctgctacat atgaggaagt 9300

attaggtggc cctccagatg gaaaggtcaa gctaaactgg atagaagaga gaccaaggat 9360

agatgtattt gtatattcat accacaaaac ttgctaattt tttttttttt tttttgagac 9420

ggagtctcgc tctgtcgccc aggctggagt gcagtggcgc aatctcggct cactgcaacc 9480

tccgcctccc gggttcacac cattctcctg cctcagcctc ctgagtagct gggactacag 9540

gcgcccgcca ccacgcccgg ctaatttttt gtatttttag tagagacggg tttcaccatg 9600

ctagccagga tggtcttgat ctcctgacct cgtcatccac ccgcctcggc ctcccaaagt 9660

gctgggatta caggcataag ccaccgcgcc tggccgtaaa gttgctatat ttctaagata 9720

agagtattta tgcagagcaa aagagatgcc aacgatcaaa ccttgagata ttcccatact 9780

tattgagtag atggaagatg aggtcagaaa aggaggaagc catgtcagta gagggtagcc 9840

ataagaaaat aacacagatt tgttatatga catcattcac aaaaatattc agtgtgattt 9900

acccctaaat caactaactt gatgtcaaaa agtaaatgta ctccagtgag taatttttct 9960

tgtgagattc aaagactcac tgaagattca ctgtgactcc aattttacta tctttctata 10020

catttctgaa tgaccaagag agctcgtaac aattatttcc tccacagaaa caaggcaaga 10080

aggaaaaaaa ctttcacatg tagaattata aatggaaaaa taaattttct agttttctta 10140

aagaccctgg tttccggtat aaagaaatgt cccagcttct tagtcacggg ccaatgggtt 10200

gttgttactg aaaaggatgg gaaacaggaa tctactattt ttgatgctgt aatgatttgt 10260

tcaggacatc acgtataccc caatctgcca acggattcct ttcctggtaa gtttggaaaa 10320

tatataataa tctagggact tatatgcaaa catcaagagt tagaaacata tctttctata 10380

ggtattacat aatgattatt cttagatttc aaaagaaaaa aattaagttt aatgatagga 10440

tatagtaata aatagcctca taagtcctta tgttaaaata atcaaggact gcaagccaga 10500

gatcagacaa acacaagttc ctgtgttaca gacagtaact caaatataag ttctaacagc 10560

acacggggtc tccgagcaca gttacattaa aaaaaagtag agtccaactg ccaaatggtt 10620

taaagaaaga cacgtttact tatgttattt ataggagact cctaggtttc taatttcatc 10680

ttcatccaca atttgcaaat aaactttaga aatctcagtg atttgtgtgt gggtacacac 10740

atgggtgtgt gtatagcagc atacttcatt accatccgaa agtggcaaac ctcaaataaa 10800

tacaatatac atggaggctt ccttccattt ttccttcctt ccttgccaca ggaacacaat 10860

ctactcaaag atattagagt ttccatgtct aggtatgatg tccataggcc gaggaaaatt 10920

aaagagtgaa ggttcaggag gaatataaga ttaaaactct taatgttaac gggcagcata 10980

tttaatgttt atgagcatgg gatcagaaca cctggcctca acttactatt ccactagttc 11040

cttaccactt aacttctttg tctcaatttc ctcttctttt aaaataggga caatagccca 11100

ccatgcaggg atgttatcaa gattaaatag ttaaaacgtg taaagcattt atcagaggat 11160

ctagcccaca gagttaactt aataaatatt aaccattatt attatcgaaa catacattct 11220

catgccttaa gattttttaa ggaactaaaa gtaagtttta gggggcttaa tgtcaaaaaa 11280

tgctaaatgg ataaatgcac ttcaactagg gaatttttta attacaactg ataataggtt 11340

taaaaagaca caaagaaaac atcttcataa tttctgaaaa tcagttcaaa caacttgcca 11400

tgttccactt aggcctggac cagtttcgag gcaactacct ccatagccgg gattataaga 11460

atccagaagc cttcaagggg aagagggtcc tcgtgattgg tctggggaat tcgggatctg 11520

acattgctgt tgagctcagc cgtctggcta cacaggtaca tgacgtaaag gttttgggaa 11580

ataaacctaa ggtagggctg tgctactaaa tcagtagcca aggcacagag gatggtactt 11640

ctatgtcaca ccacaagaga tccacctctt ctatgtggcc cttcaaatca aggaggactt 11700

gagacatcct ccatgtgaag ccaggtaatg tggcccgtgc tagtaaggaa gtacattcca 11760

ctgaatccag aagtaagtgc atgagtgcgt gtatgtacag atgagtgtgt atgtgtgtat 11820

ttcttgtttt cattttatat tctgatcacc tccaaataga ctagttcctg gtcaggctta 11880

atctttattt atttaacagt atttattata acgtatcatg caaaaagcac tgtgtttacc 11940

actctgaagt tctgaaagat atgcatgact tggtatttac taacattaat tcaatcaaca 12000

gcagatgctc aacaaatatt gggcacttac tatgcttact atgtgtcaga actatgataa 12060

actaaaaata aatgcataaa taagttagac tagttcctga cttcaagaaa gagtcaatgg 12120

atggagatgg agttgacagg tacacacaga ctatcaccag aggagatggt gagtcttcca 12180

gtagaattag gtgtggcaat agcaacacag ggaaaagaga atctaactta gcctggatga 12240

ggtcaaggaa gacttcccag aggactccaa gctaaatcat gtatcatcga tagaccctaa 12300

agaaacaaca tatttttaag aaaacaggtt ctcaataaat aaattcttaa atggatgtaa 12360

ataaaacctt aattttttaa actaaaaatt cccttcagtt atcacaaagt taaagtctat 12420

tttgcaaaga cggtaaaata gataagcagc cagactcatc tcagggctga ggcggttgcc 12480

atggtttggg ttgctcagga gaagtccttg gggtatgtgt atagggagaa ctggaaaagg 12540

caaccagaga cagagaacag aattaaatcc ttgacatctc gtcagcctaa tttcagctag 12600

agatttagct acacttttcc cacacctagt ccactatcac cagccacaac cactggggct 12660

cactggatca tctggtccct accagacttg ccatcttagt ctatgagtat gtgaagatta 12720

aaccatcaca gttgaacaca gagccctgtt gttcctagag tgatgattct aatcctttca 12780

acaactacac accagccctc aggggcagtg aaagaatcct gtctctacta gtttaaattt 12840

tagactttaa aaaaaatttt ttttatttta agttctggga tacatgtaca gaacatgcat 12900

aggtctgcac atgccatggt ggtttgctgc acctatcaac ccttcatcta gattttaagc 12960

cccacatgca ttaggtattt gtcttaatgc tctccctccc ctagccctcc atccccccga 13020

caggccttgg tgtgtgttgt tccccttcct gtgtccatgt gttctcatga ttcaactcct 13080

gcttatgagt gagaacatgc agtgttcggt tttctgttcc tgtgttagtt tgctgaggat 13140

gatggtttcc agcttcatcc atgtccctgc aaaggacatg aactcattct tttttatggc 13200

tgctagacaa cttatttaga ctcgcctttt aaaagtgttc ctacttggat attgaggaaa 13260

atgcacggaa gtgcccaaag aagtgtgttg tgtttgctta tttcttacag agtaatgctg 13320

aaatctgtgt tgcttttccc caccaggtca ttatcagtac cagaagtgct tcctgggtca 13380

tgagtcgggt ctgggatgat ggctatcctt gggatatgat gtatgttacc cgctttgcat 13440

cctttctccg gaatgtcctt ccttcattca tctctgactg gttatatgtc cagaagatga 13500

acacgtggtt taagcatgag aactatggcc tgatgccttt aaatgggtac ttaaaaatgg 13560

aaattttttt tattcaaaaa aggggggcac tcatttaatg aatttattct ctctagaact 13620

tacttttgtt gtctcattga gcctagaaac attaaactca aggtttcaca ggtgacggaa 13680

tatgcccaga gaccacgtat ggcttggaaa acttattgaa attagtccag tacagaaagg 13740

gtatggaaaa atctgaaatg gagatgacgc aggcagataa atcaccctga catgcatgat 13800

gcatttgtgg tggctacaag ctatagcata gaactttgag gactgaacaa actcaaattg 13860

gtttttggaa gaatatcttg tccgtgctta tgggtgtatg aagacatcaa taataatact 13920

tgcttctcaa gatggttgtg gtattcaata atataaaaat ataaaaattg ctttctaaat 13980

gataaagctt taaaaaaatt ggttcttctt agtctcaatt tttctaatgt gcttcaaagg 14040

agcaaataac aaaatagtgt taatcaacat gtctcagcaa gtaggaagtc tcaaaacaaa 14100

agtgcacact tcctccaccc ctgaaatgtt gacatttttg cagaaccatc aggaggcatg 14160

gaacacataa agtaatggag agtcacaact aacgtggcct gtaagattag tcagattcat 14220

ttatttactt ctttatagag acagggccca acatttacta attaggaagt cattccaggt 14280

agaagaatca gcatatcaat agaaaaaaag aatatttaag ttggtaagaa aagaaagaat 14340

tgagaaattt tatctcctgg cccatgctag ccaaaaagtt tcattgtgtt tagagaaaga 14400

tggtaagaaa aaggaggaac tgtaaatcaa aagagcaaat gccagattta ggagctaaac 14460

tgtcagtcca aagcacttat actaccaagt cttgcaggct gctataaccc tttaaaatat 14520

gttgatttta tgcatttaaa attatgttta acactgtggc ttgcttgaca gtagagggtg 14580

ggaggaggaa gagaatcaga aaaaaatact tatcaggtac tatgcttatt acccaggcga 14640

caaaattatc tatacaccaa acccctgtga cacacaattt acttatataa caaaccatgg 14700

acccccaaac ctaaaataaa agtttttaaa aattatgttt aatatagtaa gtcccatagc 14760

ttgagctggt taagattttt tatcttgtaa gagtaactat aaattatatt ttggccttgc 14820

catttagaca attaaaacat agttttagaa attcattcat tctgaaaact aagcttcctt 14880

ttggaaaggg ttccaattac cctaagtttc tggagggaga aagggggagg aaaaacaggt 14940

ttcattgtgg tctatgtttt gctaccttgt aaggtaaaag aagaggttgc aggattagat 15000

aaacagaaaa tgatgtggaa gtatagagac aaatttcagg atttacaagg tttctttgtg 15060

tctgagatac ttgcaggaaa ttccggaatc tcaaaggaaa cttaaatcaa aatgaaatat 15120

attgtcctga aaaatattat tcctagaatt ttggcaacta aaatgcaata tcaaagttgt 15180

tacacttttt tgtggacaca gctgatgaaa gaaaaccaaa catggcaata aaacttccca 15240

ccactgcaag tctgatttct ccatgtaaga caagacgtta aagttatgat aatagtgcac 15300

ttataacaac agtgcttgca tgtgccagga actgttttaa gtgctttaag gataattgat 15360

catttaattt tcacaacaac ctatgaggaa gattccatca tcatccccat tttacacata 15420

aagaaacaaa tacagaaaag taacaactag taagagatgg agctaggtta tgaacctggg 15480

ccatctgctt ccagagttgg cgttcttaac cactttagta tgtctataaa ttagttttag 15540

tctcatttag gaaaggaatt gccatgagag aagagagtca gtggcactca tgctgatgtt 15600

taagtgcttg atgttatttc aatgttatgg gctgttgcag gtatttcttg gaaatgagct 15660

atttacagca agggtgtttg cctctcattg ctgtagttcc ctgagaaaag agcctgtgtt 15720

caatgatgag ctcccatccc gcatcctgtg tggcactctg tccatcaagc ccagtgtgaa 15780

ggagttcacg gaaacctcag ctgtgtttga ggatgggacc atgtttgagg ctatcgactc 15840

tgtcatcttt gcaacaggct atgattattc ctaccccttc cttgatgaga ccatcatgaa 15900

aagcagaaac aatgaggtta ccttgtttaa aggcatcttc cccccactaa tggagaagcc 15960

aaccttggct gtgattggct tggttcagtc ccttggagct gccatcccca cagcagacct 16020

gcaagcctgg tgggctgcta aagtatttgc aagtaggtgg gccattctgt ctttcattca 16080

ttttatcaat gaacatttac tgaacacctg ctatatgcaa agcactgtgc tagggataca 16140

atgagaacaa gacaaacatg ttccttgacc tctcaaggct taaaatgggg tgtgggggat 16200

gccataatag gggaaatttg ggggggttct agtgagggga gttggactgt tgcacagagc 16260

aaacagtata caggaagtca taaaggtgag ggaaagcatg aaatgtgtaa ggacccagaa 16320

acattttggt ggaagggaat ataaagcaga ggcagggagt ggcaagaaat ataggtttat 16380

aagccacgtt aaagagctta aacttctcat agggattaag gacttcgcaa gattttaagc 16440

aagaaaaaaa tagcagagga taactgcaat gtcaggctac attataaaga ttggaagggc 16500

cctggtgagg gttggaggtg tgccagaaac ctcactggtg tcaacttctg tcagaataac 16560

aaagtcaggc cactctgatt ctcatgacaa tcttcttctt ctctccctct actctagacc 16620

tcatggtctc caggggctac aagtatgctt atgtgaggaa atcaagaata tgaggattac 16680

atggagaaag gcaatgtctc aaatatatta atttactcca gtcatactga atattatcat 16740

tattattgaa aagtgttctt ttattcaggt attctccaaa atattgacca atataggtat 16800

aacttaccta acataactaa tccataaaaa cttacactat tggtaattaa caaaccatta 16860

caatcatgga atatatgtat atatattgtc taaaactttg tagataaata aatttctatt 16920

tcaaatacac catgaaagat catcatttaa ataaacccca tcatgaaatc ttttgtaaag 16980

gtgctccctg caaaatactt ctattgcctt tttccttcga aaggcacaac aatgccaaga 17040

gcctggggta ttatgagaag actggatata gttcataaac ctaagaaatt tacatgaagc 17100

aaatggtatc atttatttat tcagcaaata cttactgaac acctactatg tgtcaggctc 17160

taacctggca cttaggacac aacaacaaac gaagcagaac aaaattctgg cctcttactt 17220

tctagcaggg tgtccagcca atatcaatca tagggtacta ccaggttgac ataagacact 17280

aacgatgact gggaaatatt catgcactgc aaattttaga gtaactttct tccactgtta 17340

caaaggcaaa taagctacca tcaccagtta aaagaagttg cattgatgta gtgaaattca 17400

caaaaagcta aaacttgtct gctgcccctt aaaacacctt gcatagttgc agaagatgtt 17460

taaaatccta tgcttccttc cattacctca tttaaaatgg cagaaacctt aaagggaact 17520

gttttaccag attctttctt cagagaagtt ttaggaaaag gatacagaaa aaaaaggaag 17580

aaattattaa gctattatat gcatgaagtg tactgagcac atatgttgag gattaggtcc 17640

tctataatgt taccgaaata agagactgag tgatttgaag ctacaaatgt ctctgctgtc 17700

actatctcac tacaggccag cttttccaat tcccaaaggt tcattaactt ttcagatctt 17760

tgtttctatg aactggtatt ttgctaaaga tatcaaagac atctccagct cctcttaata 17820

caaaagtttt caggaataca gtttataaaa accaaatgat ttccatcata tgtcattata 17880

tatttctgat ttgtgttttt caatattttt ctcttcattt cttttctaga ctcatgtacc 17940

ctgccaacca cgaatgaaat gatggatgac actgatgaga aaatggggaa aaaactcaag 18000

tggtaagcag ctaactgtac ttgctaatag agcaagttcc taaaatgtgc ctttatgtgt 18060

agaaaaacat taatatgctt taatattgtc attagtcaga gtttacattt tctgaacact 18120

tgcaataatc aaaaaatgtt tagatagtaa acagtcatca cacttctctt gtgtaactca 18180

agaatagagg ttttctatca gggataattt tgccctccag gtgacatatg gcaaaatctg 18240

gagacacttt tggtcattgt gagtggagag ggcatgctat cagcatctta tgagtagaga 18300

acagggattc tgctaaccat ccaacaatgc agagcacagt tcaccaaaac aattatctgg 18360

ctcaaaatgt caatagtgct gaggttaaga aacaactcta taaatgacta cagttgacct 18420

ttgaacaaca caggtttgaa ttatatgggt ccacttatac atggattttt tcaattaaca 18480

taatgcagat tgggcatggt ggctcacgcc tgtaatccca gcactttgtg aggctgaggc 18540

gggcggatta cctgaggtca ggagttcgag accagtctgg ccaacatggt aaaaccctgt 18600

ctctactaaa aatacaaaaa aaattagtcg agtgtggtgg tgtgcacctg taatcccagc 18660

tactcgggag gctgaggcag gggaattgct tgaatcaggg aggtggaggt tgcagtgagc 18720

caagatcgcg ccactgcact ctagcctagg tgacagagtg agactccatc tcacaaaaaa 18780

aaaaaaaaaa aatgcaattt tttggagatt tgcagcaatt taaaaactca aggccaggcg 18840

cggtggctca cgcctgtaat cccagcactt cgagaggccg aggcgggtgg atcatgagtt 18900

caggagatca agaccatcct ggctaacatg gtgaaacccc gtctctacta aaaatacacc 18960

aaaattagcc gggcgtggtg gcgggtgcct gtagtcccag ctactcggga cgctgaggca 19020

ggagaatggc gtgaacccag gaggcggact tgcagtgagc ccagattgtg ccactgcact 19080

ccagtctggg caacagagtg agactccgtc tcaaaaaaaa aaaacctcaa agatgaattg 19140

tgtagcctag aaatatttta aaaaattaag aaaaagatgc catgtataaa atatttgtag 19200

atactagtct attttatcat ttactaccat aaaatataca caaatctatt attaaaaatt 19260

aaaatttatc aaaactaaat gcatacaaac tcttagacta tacatggcac cattcatagt 19320

caacagaaat gtaaacaaac ataaagatgc aatattgtca taactgcata aaatatagca 19380

cataatgtgc tagtataata attttgcagt cacctcttgt tggtattgca gtgagctcaa 19440

gtgttttgag tatctactta aaatgctgtg tgacattagt cattttcacc tgagcagttc 19500

atatctccag taaattctgc ctcacagtaa aaagtgatct ctcaaggttc tcacatattt 19560

ttatcatgtt tagtgcaata ccttaagcct ttaataacac catgggctcc atatgaagtg 19620

tcattaatga tgttggaagt gctcccaaga agcagagaaa agttatgaca ttataataaa 19680

aaaattgagt tgcttaatgt atactataca ttgaggtctg cagctatagt tgcccaccat 19740

ttcaagataa atgaatccag tgcaactatg ccagcaggca tgaaatcttg cactttttgt 19800

aaaatatctt tttattttgg attgaaaatg cagcttttta tgtgggtgca ggattgctat 19860

aaggaagtat acatatagac tctaatataa tttgagaaaa agtgaagtta ttatatgaca 19920

aagcaaaagg aaggtgaagg atctagagct ggaaaagtta atgccagcaa aggatgattt 19980

gattacatca gaaagagttt ggcttcaaaa atgtcaagat aacaggagac acgcatgctg 20040

ccaaccaaga agaaggagat gaattcccag atgtcattat gaaaatcatt gaggagaaag 20100

gatttctgcc tgaacagatt tttaacacag acaaaagtgc cctattctgg aaaaaaaaaa 20160

aaaaaaaaag ccacaaaggc catttattaa taaggagcag aagtgagcac caagattagg 20220

caggaaagaa taagctaact actgttttgt gcaaatgcag tcagatttat gatcaggatg 20280

gcccctacct atgaagctac cccctcaaac cttgaaggga aaagatgaat atcagcttcc 20340

tatcttttgg ttatacaaga ccctttttct ggattagctc tgtcaatgct ttgtccctga 20400

agtcagaaag tccttgccaa taagagactg ccttttaaag tttttttgat atagacaatg 20460

cccctgacca cccagaaccc catgagttca acatggaagg catcgaagta gtctaatttc 20520

ccccaaacac aacattctaa ttcagccttt atatcaggga gtcataagga cctttaaggc 20580

tcatcacata ccatactcta tggaaaagat agtcaatgct gtggaagata acccaacaga 20640

gagaacatca tgaaagtctg gaaggattat accattgaag atgccctaat tgttatagaa 20700

aaagccatga aagccatcaa tcctaaaaca acatatttct cctggagaaa actatgtcca 20760

gatgttatat atgacttcag aggatttaca acagaccagt cacagaaatc atgaaaaaga 20820

ttatggatat ggcaaaataa aaaggtgagg gtgaagggtt tcaagatatg gatcatggag 20880

aaattcaaca gctaatagac accactaata gacactttta attccacact agaggaacta 20940

aaagatgact tgatggagat gagtccttcc aaagcagtgc cagatgagaa cgaagacata 21000

gaaaaagcca tgccagaaat aaattgacat tagatcatct ggcagacagg ttccagttat 21060

ttaagacttc ttttgacttc ttttatataa catggaccct tctatgatac aggcactgaa 21120

actaaagcaa atgatagagg aaggattact actatataga aaatttttag agaaataaaa 21180

aagcaaagtc agacagaaat tataatatat ttccataatt acaccaatgg gcctgcctct 21240

cctgccccca attctacctc ctccatctct tccgcttctg ccaggcctga aacagcaaga 21300

ccaacccctt ctgtttctcc tcctactcct cagcctactc aacataaaga tgataaggat 21360

gaagacattt atgataaacc acttccactt aatgaatagt aaacatattt tttcttcctc 21420

ataattttct taataacatt ttcttttctc catattactt tattgtaaga atagtattta 21480

atacgtatga catataaaat atgtgttaat caactgtttg cattattggt aaggcttcca 21540

gtcaacaggt tattaagagt taagtatttg gggagtcaaa agttatacat ggatttttga 21600

ctgcaagagg gctcaatgcc cctaacccct cagttcttcc aaagtcaact gatataggaa 21660

gtttctttac tttttcaagc atttaacatt gcattgatat gtcaacctaa aggaaaacac 21720

tgaggcaaat ttaatataaa aagagaattg atttgggcca agtttgagga ctgcaaccca 21780

ggagcacaga gtcaaattgc cctgaatatg cactccgttg gcagcagtta caagtaggtt 21840

ttttaaagga aatacaaaag agtcaacttc taagttgttt accaagaact tacattaaaa 21900

ttatataagc tattgattgg ctatatactg ttcttcgtat cacaaattct acgaacatga 21960

agatgatgag tgagacagct agtcaggaat aaaaatgcct tttaacaatt gccgccaggc 22020

ttggtagagg gcagcatgac aagtcccata cacgtggctc tctcagcttg ataaattttg 22080

catacctcac atagtgcaaa ctactctgag ctatttttct tctctcacat tgaatgccac 22140

aatgtagtca cccattcagg gcctagagaa gaaaagaaat ggaaccctca gattcaacaa 22200

aacctctcct gcacaacttc agccagttga cgaacaactt gcagagttgg gcacttttat 22260

gtgctaacaa ttcatgcagc ttgataccct ttcctttaga gcccagtaga aataaaaatg 22320

aggaaataga gaggttaaaa tgttcatctt attgcttaaa tgataagctg ctcttcagag 22380

tttcaaaaag caaattacac catattccaa ctaaaagaac tatagaggcg gaaaggaggt 22440

gatctctttt ctctctgtca taaaaggtaa tggccaacac ccctataaca aaagacaggt 22500

taacaagaga aaacgtgaca gatttattac gtgcacatgt gtgcatgaga gccttacaaa 22560

acatgaactc aaaggagggc cagatcattc atgtttaaat attctcttca ctggggttag 22620

gggagatgga agtgtaaaag taaatgattt ttcagaggaa attaataagt ccaaagaaca 22680

cagattagac caagtttctc tgggctttgg gggaggtgta atcacccaac agattcatct 22740

tgctcactgc ccagaaaagc tgatgccctg agaacagcag gtttttccaa tagagagagt 22800

ttaataaaca cacagctgtc agaggcattt gaaccagagt gactccatct tgaatagggg 22860

ctgggtaaaa tgaggctgag acccactagg ctgcattccc aggaggttag gcattcttag 22920

tcacaggatg agaaaagagg ccagcacaag attcaggtca caaagacctg gctgataaaa 22980

caggattcag taacgaagct ggccaaaacc caccaaaacc aagatgatga aaaaagtgac 23040

ctctggtcgt cctcactact cattatatgc tgattataat gaattagcat gctagaagac 23100

actcccacca gcactgtgat agtttacaaa tgccatggca atatcaggaa gttaccctat 23160

atagtctaaa aaggggagga accctcagtt ctgggaactg cccatttctt tcctgtaaaa 23220

cttatgaata atccacccct tgtttaacat gtaatcaaga agtaactata agtatactca 23280

gttgagcagc ccatgccact gctctgccta tgaagtagcc attcttttat tcctttactt 23340

tcttaataaa ctcgctttca ctttatggac tggccctgaa ttctttcttg tgagaggtcc 23400

aagaaccctc ttttggggtc tggccagcta aacggaagga caggagttta ttactactca 23460

aatcagcctc catgaaaatt cagaggctag atttttttaa ggatagtttg gtagtcaggg 23520

gctagggaat ggggaatgct gattggttgg gtcagggatg aaaccatagg gagtcaaagc 23580

ttgtcttctg gtcttcctgg gaggagacca catgacaaga tgaaccagtt taccagtctg 23640

ggtagtgcca gccggcccat cagaatgcag ggtctgaaaa atatcttgag caccaatggt 23700

aggttttata atggtgatgt tatccatagg agcaattggg gacttctgac tgcatgactc 23760

ctgagcccta atttcttatc ttgtggctaa tttgttagtt ctacaaaagc agtctgatct 23820

ccaagcaagg agggggtttg ttttgggaaa gggctgttac catctttgtt tcaaagttaa 23880

actgtaaact aaatgtctcc catagttagc ttggcctatg ctcaggaatg aataatggca 23940

gcttggagat tagaagaaag atggagtaat tacatttttt tttcacattt ttttcactgt 24000

cacaattttt ttaaaggtga tttcagaggt aacatcacag gacatgggag actaaaggga 24060

ggaaagtatg tcaaacaaag gctgtcctgt tctgcagacg aaacctcaca gaaagcaact 24120

ctcagagtca gtagcctatg atgaaagttt ctctgtcaga cattcagcag tgcctgactc 24180

tcagtctctc tctcctgcaa gttaatcttt cctagagtgg gcaagggagg cctccgagaa 24240

agcctagttt ccatcttctg tttacttcct tttattttct ccacagataa aaatctcctt 24300

cacaaaaggc agcttttcag ggctgtttct gtctgcaggc cctctgaata gccatctcaa 24360

aatctgtcaa cgaagtgtat attttgcagt aaaatatttt ttgttttctt tagtatgaaa 24420

caatttatat tattagatta caggagtatt aaaaccatcc atgatctcac ttttaaacaa 24480

accaatctga aagtctaaca ttggggcaga ttctaagcaa tgtcttataa agaataatta 24540

tgtgttaatg agtaaactaa gttaattagt ctccttaaac cagagggtca gtttactcca 24600

ggccacatgg tcaaaggcaa aagtccaaca ttacatcaaa ctcaaataga gattaggaag 24660

gaggagaaaa gcagctcact tagctaaaga aaaaacaata aattcaattt tgtggaaaag 24720

gagggcataa atggaggtgc tatctaaaat gttatttttc tgaaagaaaa aataagaaat 24780

taatgctcct atttgcaact gtaacactta ttccagtatg ttctcttctt tcttcatgtt 24840

tggccagagc cagactttgc agacagatta catcacatat gtggatgagc tgggctcttt 24900

cataggggcc aagcctaaca taccatggct cttcctgaca gatccccgcc tggccctgga 24960

ggtgtacttt ggcccttgca gcccatacca gtttcgactg atgggaccag ggaagtggga 25020

tggggccaga aatgccatcc tgacccagtg gaaccggaca gtgaagccaa ccaggacaag 25080

agttgtcagt gaagttcagc gaccccatcc cttttacaat ttgcttaaaa tgctttcatt 25140

cccattactc cttctggctg ttacacttac attttattaa tgagaaagtc tttgaggtct 25200

caaaattcag catagaagtg taatcacaca atacaacaca caccacacat acacacacac 25260

aatcacaaca tagttcctct ctcctttcct gaagatatga aaatcagtct tggcccattt 25320

gaattaaagt ataagtaaaa tggaaaatac tcagcctctc tctctctgtt gggaatctgt 25380

tctctaaaag gcttttcaca tgctgaattg gcaaatttgg ggatgcttaa gataagacag 25440

gaagttgaat aagcatgagc acag 25464

5

gcaatcatga gcaagagggt tggcatcatc ggagctggag tcagtggctt ggctgccata 60

tggtgctgtc tggaggaggg gctggagccc acttgctttg aaaggagcga tgatgttgga 120

ggcctgtgga aattctcaga ccacacagaa gaaggcagag ccagcattta ccagtctgta 180

ttcacaaact cttccaaaga aatgatgtgc tttccagact tcccttatcc ggatgattac 240

ccaaactata tacaccacag caagctccag gaatatataa agacatatgc tcaaaagaag 300

gaacttttaa gatacatana gtttgagacc ctggtttccg gtataaagaa atgtcccagc 360

ttcttagtca cgggccaatg ggttgttgtt actgaaaagg atgggaaaca ggaatctact 420

atttttgatg ctgtaatgat ttgttcagga catcacgtat accccaatct gccaacggat 480

tcctttcctg gcctggacca gtttcgaggc aactacctcc atagccggga ttataagaat 540

ccagaagcct tcaaggggaa gagggtcctc gtgattggtc tggggaattc gggatctgac 600

attgctgttg agctcagccg tctggctaca caggtcatta tcagtaccag aagtgcttcc 660

tgggtcatga gtcgggtctg ggatgatggc tatccttggg atatgatgta tgttacccgc 720

tttgcatcct ttctccggaa tgtccttcct tcattcatct ctgactggtt atatgtccag 780

aagatgaaca cgtggtttaa gcatgagaac tatggcctga tgcctttaaa tggttccctg 840

agaaaagagc ctgtgttcaa tgatgagctc ccatcccgca tcctgtgtgg cactctgtcc 900

atcaagccca gtgtgaagga gttcacggaa acctcagctg tgtttgagga tgggaccatg 960

tttgaggcta tcgactctgt catctttgca acaggctatg attattccta ccccttcctt 1020

gatgagacca tcatgaaaag cagaaacaat gaggttacct tgtttaaagg catcttcccc 1080

ccactaatgg agaagccaac cttggctgtg attggcttgg ttcagtccct tggagctgcc 1140

atccccacag cagacctgca agcctggtgg gctgctaaag tatttgcaaa ctcatgtacc 1200

ctgccaacca cgaatgaaat gatggatgac actgatgaga aaatggggaa aaaactcaag 1260

tggtttggcc agagccagac tttgcagaca gattacatca catatgtgga tgagctgggc 1320

tctttcatag gggccaagcc taacatacca tggctcttcc tgacagatcc ccgcctggcc 1380

ctggaggtgt actttggccc ttgcagccca taccagtttc gactgatggg accagggaag 1440

tgggatgggg ccagaaatgc catcctgacc cagtggaacc ggacagtgaa gccaaccagg 1500

acaagagttg tcagtgaagt tcagcgaccc catccctttt acaatttgct taaaatgctt 1560

tcattcccat tactccttct ggctgttaca cttacatttt attaa 1605

6

532

Homo sapiens

6

Met Ser Lys Arg Val Gly Ile Ile Gly Ala Gly Val Ser Gly Leu Ala

1 5 10 15

Ala Ile Trp Cys Cys Leu Glu Glu Gly Leu Glu Pro Thr Cys Phe Glu

20 25 30

Arg Ser Asp Asp Val Gly Gly Leu Trp Lys Phe Ser Asp His Thr Glu

35 40 45

Glu Gly Arg Ala Ser Ile Tyr Gln Ser Val Phe Thr Asn Ser Ser Lys

50 55 60

Glu Met Met Cys Phe Pro Asp Phe Pro Tyr Pro Asp Asp Tyr Pro Asn

65 70 75 80

Tyr Ile His His Ser Lys Leu Gln Glu Tyr Ile Lys Thr Tyr Ala Gln

85 90 95

Lys Lys Glu Leu Leu Arg Tyr Ile Gln Phe Glu Thr Leu Val Ser Gly

100 105 110

Ile Lys Lys Cys Pro Ser Phe Leu Val Thr Gly Gln Trp Val Val Val

115 120 125

Thr Glu Lys Asp Gly Lys Gln Glu Ser Thr Ile Phe Asp Ala Val Met

130 135 140

Ile Cys Ser Gly His His Val Tyr Pro Asn Leu Pro Thr Asp Ser Phe

145 150 155 160

Pro Gly Leu Asp Gln Phe Arg Gly Asn Tyr Leu His Ser Arg Asp Tyr

165 170 175

Lys Asn Pro Glu Ala Phe Lys Gly Lys Arg Val Leu Val Ile Gly Leu

180 185 190

Gly Asn Ser Gly Ser Asp Ile Ala Val Glu Leu Ser Arg Leu Ala Thr

195 200 205

Gln Val Ile Ile Ser Thr Arg Ser Ala Ser Trp Val Met Ser Arg Val

210 215 220

Trp Asp Asp Gly Tyr Pro Trp Asp Met Met Tyr Val Thr Arg Phe Ala

225 230 235 240

Ser Phe Leu Arg Asn Val Leu Pro Ser Phe Ile Ser Asp Trp Leu Tyr

245 250 255

Val Gln Lys Met Asn Thr Trp Phe Lys His Glu Asn Tyr Gly Leu Met

260 265 270

Pro Leu Asn Gly Ser Leu Arg Lys Glu Pro Val Phe Asn Asp Glu Leu

275 280 285

Pro Ser Arg Ile Leu Cys Gly Thr Leu Ser Ile Lys Pro Ser Val Lys

290 295 300

Glu Phe Thr Glu Thr Ser Ala Val Phe Glu Asp Gly Thr Met Phe Glu

305 310 315 320

Ala Ile Asp Ser Val Ile Phe Ala Thr Gly Tyr Asp Tyr Ser Tyr Pro

325 330 335

Phe Leu Asp Glu Thr Ile Met Lys Ser Arg Asn Asn Glu Val Thr Leu

340 345 350

Phe Lys Gly Ile Phe Pro Pro Leu Met Glu Lys Pro Thr Leu Ala Val

355 360 365

Ile Gly Leu Val Gln Ser Leu Gly Ala Ala Ile Pro Thr Ala Asp Leu

370 375 380

Gln Ala Trp Trp Ala Ala Lys Val Phe Ala Asn Ser Cys Thr Leu Pro

385 390 395 400

Thr Thr Asn Glu Met Met Asp Asp Thr Asp Glu Lys Met Gly Lys Lys

405 410 415

Leu Lys Trp Phe Gly Gln Ser Gln Thr Leu Gln Thr Asp Tyr Ile Thr

420 425 430

Tyr Val Asp Glu Leu Gly Ser Phe Ile Gly Ala Lys Pro Asn Ile Pro

435 440 445

Trp Leu Phe Leu Thr Asp Pro Arg Leu Ala Leu Glu Val Tyr Phe Gly

450 455 460

Pro Cys Ser Pro Tyr Gln Phe Arg Leu Met Gly Pro Gly Lys Trp Asp

465 470 475 480

Gly Ala Arg Asn Ala Ile Leu Thr Gln Trp Asn Arg Thr Val Lys Pro

485 490 495

Thr Arg Thr Arg Val Val Ser Glu Val Gln Arg Pro His Pro Phe Tyr

500 505 510

Asn Leu Leu Lys Met Leu Ser Phe Pro Leu Leu Leu Leu Ala Val Thr

515 520 525

Leu Thr Phe Tyr

530

7

21

Artificial Sequence

oligonucleotide

7

tcacatagag tgctatgggg g 21

8

25

Artificial Sequence

oligonucleotide

8

cttaggaaga agataaaaat gcaac 25

9

23

Artificial Sequence

oligonucleotide

9

aatgtccatc atcatagttc tct 23

10

23

Artificial Sequence

oligonucleotide

10

taggcttgtg tagcctgccc tca 23

11

16

Artificial Sequence

oligonucleotide

11

cctcagagag aactat 16

12

16

Artificial Sequence

oligonucleotide

12

ggagtctctc ttgata 16

13

16

Artificial Sequence

oligonucleotide

13

cctcaaagag aactat 16

14

16

Artificial Sequence

oligonucleotide

14

ggagtttctc ttgata 16