US20060154295A1

US20060154295A1 - Nucleotide sequence which encodes a flavin monooxygenase, the corresponding protein and their uses in the spheres of diagnosis and therapy

Info

Publication number: US20060154295A1
Application number: US11/378,840
Authority: US
Inventors: Marta Blumenfeld; Ilia Tchoumakov; Henri-Jean Garchon; Jean-Francois Bach
Original assignee: Serono Genetics Institute SA
Current assignee: Merck Biodevelopment SAS
Priority date: 1996-12-06
Filing date: 2006-03-17
Publication date: 2006-07-13
Also published as: DE69737293T2; US20030203463A1; WO1998024914A1; PT941341E; AU5327198A; JP4062638B2; FR2756845B1; FR2756845A1; CY2605B2; US7037709B2; EP0941341A1; ATE352635T1; DE69737293D1; ES2279552T3; CA2274011A1; DK0941341T3; US6551792B1; EP0941341B1; AU746293B2; JP2001505430A

Abstract

The present invention concerns, in particular, human flavin-containing monooxygenase 2 (hFMO2), and another human enzyme of the FMO, hFMOx family, their nucleotide and polypeptide sequences. The present invention also concerns vectors for cloning and/or expression containing said nucleotide sequences and cells transformed by these vectors and method for preparing said polypeptides. The invention further concerns methods for selecting compounds and of diagnosing predisposition to pathologies and/or deficiencies related to FMO's and pharmaceutical compositions containing said compounds for treating and/or preventing these pathologies.

Description

RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No. 10/374,228, filed Dec. 25, 2003, which is a divisional of U.S. application Ser. No. 09/326,480, filed Jun. 4, 1999 (the disclosure of which is incorporated herein in its entirety), now U.S. Pat. No. 6,551,792, which is a 371 application of PCT application Serial Number PCT/FR97/02226, 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. FMO3 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 FMO3. 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.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: Analysis of the segregation of the G.1263mac.A pblymorphism 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.

BRIEF DESCRIPTION OF THE TABLES

Table 1 depicts primer sequences which can be used 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 strucuture of the gene encoding hFMOx. The positions where the exons being 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/Genethon 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 (Table 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 used 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.

EXAMPLES

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 mocroconcentrators 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 MgCl₂, 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:
Final volume 50 μl
Genomic DNA 100 ng
MgCl2 2 mM
(for each) dNTP 200 μM
(for each) primer 7.5 pmol
AmpliTaq Gold DNA polymerase (Perkin) 1 unit
PCR buffer 1×
(10×=0.1 M Tris HCl, pH 8.3, 0.5 M KCl)
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 (G/G), 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.

TABLE 1


Sequences of the primers employed for
amplifying the exon region which was
derived from BAC 123H04M and which
contains a heterozygosity site which is
linked to juvenile POAG

Locus of the	FMO2/Exon 8
fragment:

Size of the amplified	420
fragment:

Primers:
Upstream PU	5′ TCACATAGAGTGCTATGGGGG
(SEQ ID No. 7):

Downstream RP	5′ CTTAGGAAGAAGATAAAAATGCAAC
(SEQ ID No. 8):

TABLE 2


Examples of primers for detecting the
G.1263mac.A mutation by “Single
Nucleotide Primer Extension”

a)	SEQ ID No. 9:	5′ AATGTCCATCATCATAGTTCTCT 3′
		(antisense)

and/or

b)	SEQ ID No. 10:	5′ TAGGCTTGTGTAGCCTGCCCTCA 3′
		(sense)

TABLE 3


Identification of the G.1263mac.A mutation by
RFLP




	DdeI site (C TNAG)
	5′ CCCTCAaAGAGAA 3′ “mutant”
	No cleavage

TABLE 4


Example of probes for detecting the G.1263mac.A.
mutation by the ASO technique








	its complementary strand
	SEQ ID No. 14: 3′ GGAGTTTCTCTTGATA 5′

TABLE 5


Sequences of the primers employed for
sequencing the amplification fragments
derived from the genomic DNA

	PU	5′ TGTAAAACGACGGCCAGT

	RP	5′ CAGGAAACAGCTATGACC

TABLE 6


Lod score between the G.1263mac.A polymorphism and the juvenile
POAG in the studied family as a function of the frequency
of the two alleles in the general population

Frequency of the	Θ (recombination
rare (A) allele	rate)	Lod score

0.01	0	2.07
0.001	0	2.10
0.0001	0	2.10
0.00001	0	2.10

TABLE 7A


	Position in the	Position in the
FMO2	gene (SEQ ID NO: 1)	mRNA (SEQ ID NO: 2)

Exon 1	2001-2056	1-56
Exon 2	2405-2542	57-194
Exon 3	10026-10214	195-383
Exon 4	13341-13503	384-546
Exon 5	16036-16178	547-689
Exon 6	20558-20757	690-889
Exon 7	21972-22327	890-1245
Exon 8	24411-24483	1246-1318
Exon 9	25487-25899	1319-1731
ATG	2411-2413	63-65
Stop	25836-25838	1668-1670

TABLE 7B


	Position in the	Position in the
FMOx	gene (SEQ ID NO: 4)	mRNA (SEQ ID NO: 5)

Exon 1	2001-2138	1-138
Exon 2	6961-7149	139-327
Exon 3	10144-10306	328-490
Exon 4	11413-11555	491-633
Exon 5	13347-13546	634-833
Exon 6	15697-16052	834-1189
Exon 7	17930-18002	1190-1262
Exon 8	24838-25180	1263-1605
CDS	2006-25180	6-1605

TABLE 8


FMO2 Homology Between Macaque and Human

Length	% Amino Acid	% DNA
(nucleotide)	Homology	Homology

Exon 1 (5′UTR)	64	—	95.3
Exon 2	137	100	96.5
Exon 3	188	98	96.8
Exon 4	162	96.7	96.9
Exon 5	142	95.8	96.5
Exon 6	199	95.4	97
Exon 7	355	98.3	97.7
Exon 8	72	96	97.2
Exon 9 (3′UTR)	413	93	95
Total		96	96.7

TABLE 9


Variations Between Human and Macaque FMO2

Position	Macaque	Human
Macaque mRNA	Nucleotide	Nucleotide	Amino Acid

56	A	G	Non-coding
71	A	G	—
83	C	T	—
104	G	A	—
197	G	T	Lys −> Asn
218	C	T	—
266	T	C	—
284	C	T	—
344	C	T	—
360	T	C	—
404	G	A	—
455	T	C	—
482	T	C	—
499	C	G	Ser −> Thr
510	T	A	Ile −> Phe
548	C	G	Ile −> Met
604	T	C	Ser −> Phe
629	C	T	—
650	C	A	—
676	G	A	Asn −> Ser
725	T	C	—
729	G	A	Val −> Ala
743	T	C	Arg −> Gln
758	G	A	—
811	T	C	—
844	A	G	—
995	T	C	—
1085	T	C	Glu −> Asp
1121	G	A	Phe −> Leu
1133	A	C	—
1145	G	C	—
1155	T	C	Ser −> His
1157	T	C	Ser −> His
1160	A	G	—
1251	C	A	—
1252	A	G	Tyr −> Phe
1370	T	C	—
1448	G	C	—
1450	T	A	—
1473	C	N	—
1484	A	G	—
1486	C	T	—
1509	G	N	—
1510	C	N	—
1514	G	A	—
1516	G	A	—
1535	A	G	—
1541	G	A	—
1556	A	C	—
1567	T	C	—
1590	C	T	—
1598	C	T	—
1623	G	C	—
1646	C	T	—
1677	T	C	—
1678	G	A	—

REFERENCES

The following publications are incorporated herein by reference in their entireties:

Allen J. B., Walberg M. W., Edwards M. C. & Elledge S. J. Finding prospective partners in the library: the two hybrid system and phage display find a match. TIBS 20: 511-516 (1995).
Altschul, Stephen F., Gish W., Miller W., Myers E. W., & Lipman D. J. Basic local alignment search tool. J. Mol. Biol. 215:403-10 (1990).
Bairoch A. & Apweiler R. The SWISS-PROT protein sequence data bank and its new supplement TREMBL. Nucleic Acids Res. 24: 21-25 (1996).
Belmouden A., Adam M. F., Dupont de Dinechin S., Brézin A. P., Rigault P., Chumakov I., Bach J-F., & Garchon H-J., 1996, Recombinational and physical mapping of the locus for primary open-angle glaucoma (GLCLA) on chromosome 1q23-q25. Genomics, sous presse.
Benson D. A., Boguski M., Lipman D. J. & Ostell J. GenBank. Nucleic Acids Res. 24: 1-5 (1996).
Bonfield J. K., Smith K. F. & Staden R. A new DNA sequence assembly program. Nucleic Acids Res. 23: 4992-9 (1995).
Buckholz R. G. Yeast Systems for the Expression of Heterologous Gene Products. Curr. Op. Biotechnology 4: 538-542 (1993).
Cashman J. R., Park, B. P., Berkman, C. E. & Cashman, L. E. Rôle of hepatic flavin-monoxygenase 3 in drug and chemical metabolism in adult humans. Chemico-Biological Interactions 96: 33-46 (1995).
Carter B. J. Adeno-Associated virus vectors. Curr. Op. Biotechnology 3: 533-539 (1993).
Cherif D., Julier C., Delattre O., Derré J. Lathrop G. M., & Berger R.: Simultaneous localization of cosmids and chromosome R-banding by fluorescence microscopy—Applications to regional mapping of chromosome 11. Proc. Natl. Acad. Sci. USA. 87: 6639-6643 (1990).
Chumakov I., Rigault P., Guillou S., Ougen P., Billault A., Guasconi G., Gervy P., Le Gall I., Soularue P., Grinas P. et al. Continuum of overlapping clones spanning the entire human chromosome 21q. Nature 359: 380-386 (1992).
Chumakov I. M., Rignault P., Le Gall I. et al. A YAC contig map of the human genome. Nature 377 supplt: 175-183 (1995).
Compton J. Nucleic Acid Sequence-Based Amplification. Nature 350: 91-92 (1991).
Danos O., Moullier P. & Heard J. M. Réimplantation de cellules génétiquement modifiees dans des néo-organes vascularisés (Reimplantation of genetically modified cells in vascularized neoorgans). Médecine/Sciences 9:62-64 (1993).
Edwards C. P. et Aruffo A. Current applications of COS cell based transient expression systems. Curr. Op. Biotechnology 4: 558-563 (1993).
Epstein A.: Les vecteurs herpétiques pour le transfert de gènes (Herpesvirus vectors for transferring genes)—Médecine/Sciences 8: 902-911 (1992).
George D. G., Barker W. C., Mewes H. W, Pfeiffer F. & Tsugita A. The PIR-International Protein Sequence Database. Nucleic Acids Res. 24: 17-20 (1996).
Guatelli J. C. et al. Isothermal in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proc. Natl. Acad. Sci. USA 87: 1874-1878 (1990).
Hillier L. & Green P. OSP: a computer program for choosing PCR and DNA sequencing primers. PCR Methods Appl. 1: 124-8 (1991).
Hines et al., Toxicol. Appl. Pharmacol. 125, 1-6 (1994).
Landegren U., Kaiser R., Sanders J. & Hood L. A ligase-mediated gene detection technique. Science 241: 1077-1080 (1988).
Lawton M. P., Cashman J. R., Cresteil T., Dolphin C. T., Elfarra A. A., Hines R. N., Hodgson E., Kimura T., Ozols J., Phillips I. R., Philpot R. M., Poulsen L. L., Rettie A. E., Shephard E. A., Williams D. E., & Ziegler D. M.: A nomenclature for the mammalian flavin-containing monooxygenase gene family based on amino acid sequence identities. Arch. Biochem. Biophys. 308:1, 254-257 (1994).
Luban J. & Goff S. P. The yeast two-hybrid system for studying protein-protein interactions. Current Op. Biotechnology 1995, 6:59-64.
Luckow V. A. Baculovirus systems for the expression of human gene products. Curr. Op. Biotechnology 4: 564-572 (1993).
Olins P. O. et Lee S. C. Recent advances in heterologous gene expression in E. coli. Curr. Op. Biotechnology 4:520-525 (1993).
Park, S. B. et al., Chem. Res. Toxicol. 5, 193-201 (1992).
Perricaudet M., Stratford-Perricaudet L., & Briand P.: La thérapie génique par adénovirus (Gene therapy using adenoviruses)—La Recherche 23: 471-473 (1992).
Poulsen, L. L. et al., Chem. Biol. Interact. 96, 57-73 (1995).
Rodriguez-Tome P., Stoehr P. J., Cameron G. N., & Flores T. P. The European Bioinformatics Institute (EBI) databases. Nucleic Acids Res. 24: 6-12 (1996).
Samiotaki M., Kwiatkowksi M. Parik J., & Landegren U. Dual-color detection of DNA sequence variants through ligase-mediated analysis. Genomics 20: 238-242 (1994).
Schwartzman, M. L., Masferrer, J., Dunn M. W., McGiff J. C., Abracham N. G., 1987, Curr Eye Res. 6: 623-630.
Schwartzman M. L., Balazy M., Masferrer J., Abraham, N. G., McGiff, J. C., Murphy, R. C., 1987, PNAS USA 84: 8125-8129.
Stoneking M., Hedgecock D., Higuchi R. G., Vigilant L., & Erlich H. A. Population variation of human DNA control region sequences by enzymatic amplification and sequence-specific oligonucleotide probes. Am. J. Hum. Genet. 48: 370-382 (1991).
Sunden S. L. F., Alward W. L. M., Nichols B. E., Rokhlina T. R., Nystuen A., Stone E. M. & Sheffield V. C. Fine mapping of the autosomal dominant juvenile open angle glaucoma (GLC1A) region and evaluation of candidate genes. Genome research 6: 862-869 (1996).
Syvänen A. C., Aalto-Setala K., Harju L., Kontula K., & Soderlund H. A primer-guided nucleotide incorporation assay in the genotyping of Apo E. Genomics 8: 684-692 (1990).
Temin H. M.: Retrovirus vectors for gene transfer. In Kucherlapati R., ed. Gene Transfer, New York, Plenum Press, 149-187 (1986).
Walker G. T., Fraiser M. S., Schram J. L., Little M. C., Nadeau J. G., & Malinowski D. P. Strand displacement amplification an isothermal in vitro DNA amplification technique. Nucleic Acids Res. 20: 1691-1696 (1992).
Wu D. Y., Ugozzoli L. Pal B. K., Wallace R. B. Allele-specific amplification of b-globin genomic DNA for diagnosis of sickle cell anemia. Proc. Natl. Acad. Sci. USA 86: 2757-2760 (1989).
Ziegler, D. M., Drug Metab. Rev. 19, 1-32 (1988).
Ziegler, D. M., Annu. Rev. Pharmacol. Toxicol., 33, 179-199 (1993).

Claims

1. A method of diagnosing predisposition to juvenile primary open-angle glaucoma (jPOAG) comprising analyzing a biological sample for a mutation in at least one sequence, wherein said sequence comprises SEQ ID NO: 3 and said mutation is the substitution of a lysine for a glutamic acid at position 402 of SEQ ID NO: 3 or the substitution of an A for a G in at a position corresponding to nucleotide 24433 of SEQ ID NO: 1.

2. The method according to claim 1, wherein said biological sample is genomic DNA, cDNA or mRNA.

3. The method according to claim 2, wherein all or a part of the nucleic acid sequence is analyzed for the substitution of an A for a G at a position corresponding to nucleotide 24433 of SEQ ID NO: 1.

4. The method according to claim 3, wherein said analyzing step comprises amplifying a nucleic acid sequence comprising the nucleotide corresponding to nucleotide 24433 of SEQ ID NO: 1.

5. The method according to claim 4, wherein said nucleic acid sequence comprises a span of 10, 20 or 30 nucleotides.

6. The method according to claim 2, wherein said biological sample is genomic DNA.

7. The method according to claim 2, wherein said biological sample is cDNA.

8. The method according to claim 2, wherein said biological sample is mRNA.

9. The method according to claim 3, wherein said biological sample comprises SEQ ID NO: 3.

10. The method according to claim 9, wherein said analyzing step comprises contacting a polypeptide comprising SEQ ID NO: 3 with an antibody to detect the presence of said polypeptide.

11. The method according to claim 10, wherein said antibody is labeled with a detectable molecule.