JP2003523743A - MLP genes, nucleic acids, polypeptides and uses thereof - Google Patents

Abstract

(57) [Summary] The present invention relates to a mutant MLP sequence wherein the mutation is made at the 10th base of exon 2 of the translated sequence or at position 3 of codon 112 of exon 4 of the translated sequence. Furthermore, the invention relates to a muscle-specific promoter and its use.

Description

Detailed Description of the Invention

Description: The present invention relates to nucleic acids encoding MLPs, polypeptides encoded thus, probes for these nucleic acids, methods for detecting and / or testing myocardial disease, detection kits for nucleic acids and polypeptides, regulatory nucleic acids, Vectors containing the latter, cells containing the latter, as well as agents containing the latter.

Dilated cardiomyopathy (DCM) is a diminished contractility (disruption of the systole, which is a ventricular function), and a concomitant diastolic relaxation of the left and / or right ventricles (a diastole, a ventricular function). Destruction) occurs at the same time, and is a myocardial disease of nuclear origin. Cardiac hypertrophy, diminished output fraction, and high diastolic volume due to biventricular dilation are typical. The output of the heart is reduced (anterior failure), which results in the return of blood flow from the heart (posterior failure) (Isselbacher, K. et al; Harris).
son's Principles of Internal Medicine
e (McGraw Hill, New York, 1994)).

In western industrialized countries, the incidence of DCM is about 5 / 100,000 residents / year. Therefore, it represents the most common early cardiomyopathy. In a recent study, family clusters were reported in about 30% (To
wbin, The Role of Cytoskeletal Protei
ns in Cardiomyopathy 10, 13-139 (199
8)). The cumulative cost of this disease is, for example, 100-400 in the United States.
It is estimated to have accumulated US $ 100 million, or 5 to 20 billion DM in Germany.

However, the etiology of this disease is still unclear. In about 30% of cases, enterovirus DNAs and RNAs (Pauschi) were introduced into the myocardium.
Nger, M .; et al. Circulation 99 (7), 889-
895 (1999)) or adenovirus DNAs and RNAs (Pausc)
hinger, M .; et al. Circulation 99 (10), 1
348-1354 (1999)) can be detected. A possible pathological mechanism is Badorff (Badorff, C. et al .; Nature).
Medicine 5,320-326 (1999)). Therefore, viral origin may be considered appropriate. Moreover, antibodies such as beta
Since antibodies against the receptor and adenine nucleotide translocator (ANT) were detected, the autoimmune mechanism was also examined as a pathogenic principle (Sc
hultheiss, H .; P. et al. Circ. Res. 76,64
-72 (1995)). However, finally, even in DCM, especially family clusters tend to show genetic causes. For example, mutations within the dystrophin gene are best demonstrated by Duchenne and Becker muscular dystrophy, which also results in DCM in addition to muscular dystrophy. Besides, DC
Two mutations within the myocardial actin gene have been described in a corresponding family that produces M (Olsen, T. et al .; Science 280, 750-752 (
1998)). The mutation was also found in the meta-vinculin gene and probably resulted in cardiomyopathy in the patient (Maedam, M. et al .;
Circulation 95, 17-20 (1997)). Furthermore, in Syrian hamster cardiomyopathy, a causative mutation in the delta sarcoglycan gene can be detected (Niro, V. et al .; Hum. Mol.
． Genet. 6, 601-607 (1997)). In sharp contrast to the sarcomeric proteins whose mutations clearly produce hypertrophic cardiomyopathy (HCM), mutations in cytoskeletal proteins result in DCM because these genes all together encode cytoskeletal proteins ( Towbin, J. et al .; Natu
re Medicine 5,266-267 (1999) and Chen, J. et al.
et al. J Clin Invest 103, 1483-1485 (1
999)).

Since MLPs are listed as cytoskeletal proteins, MLPs that match DCM
Knock out Immediately add to the conceptual model shown above (Arber et al.
． Cell 88, 393-403 (1997)).

In 1994, MLP was first invented as a positive regulator of myogenesis by using subtractive cloning technology (Arber, S. et al .; C.
ell 79, 221-31 (1994)), and 1997, it was known that MLP knockouts in a mouse model co-produce DCM, and gained considerable importance for heart disease (Arber et al .; Cell 88). , 3
93-403 (1997)). This was at the same time the first transgenic organism to stand out in this clinical picture. However, in the prior art it is totally unknown to date whether mutations within this gene also give rise to DCM in humans.

MLPs themselves, as the name implies, belong to the group of LIM proteins. About 1
0 years, this group is named after the beginning character of their first three elements (l in11, i slet1, m ec3), and basically the following three subgroups: 1, L
IM-kinase, ie a protein having LIM motif and kinase activity, 2, LIM homeobox gene, ie LIM protein acting as a transcription factor, and 3, “LIM only” protein, ie LIM protein carried only in the LIM motif, Have. MLPs belong to the group of "LIM only" proteins (Morgan, M. et al .; Biochem Bioph.
ys Res Com 212, 840-846 (1995)).

The protein itself consists of 194 amino acids, and LIM double zinc
It forms fingers, and in each case a glycine-rich amino acid sequence. The protein probably acts as a cytoskeleton due to its localization to the z-band, which binds to the f-actin structure (Arber, S. et al .; Gen.
e Dev 10, 289-300 (1996)).

The object of the present invention is to provide agents which result in the prophylactic treatment of dilated cardiomyopathy. The agents are individually placed (ar) in the progression of the clinical picture of dilated cardiomyopathy.
It also results in the detection of range. Finally, the agent to be provided enables the diagnosis of dilated cardiomyopathy.

Furthermore, the object of the present invention is to provide a method for the detection of myocardial disease, the examination of myocardial disease, and the detection of the arrangement with respect to the progression of myocardial disease, in which case the myocardial disease is especially dilated is there.

Finally, it is an object of the present invention to provide a kit which can be used within the framework of said method.

In another aspect, an object of the present invention is to provide regulatory nucleic acids that result in tissue-specific, particularly muscle-specific, nucleic acid expression.

Further, it is an object of the present invention to provide an agent for treating and / or preventing a disease which can be treated or prevented by tissue-specific expression.

Said object is achieved according to the invention by a nucleic acid which codes for an MLP and which comprises a sequence according to SEQ ID NO: 1 having a mutation in the 10th base of the second exon of the translated sequence.

[0015]   In a preferred embodiment, the mutation is a T to C conversion.

Furthermore, said object is achieved by a nucleic acid which encodes an MLP and which comprises a sequence according to SEQ ID No. 1 having a mutation at position 3 of codon 112 within exon 4 of the translated sequence.

[0017]   In a preferred embodiment, the mutation is an A to G conversion.

In another preferred embodiment of the nucleic acid according to the invention, the nucleic acid comprises only the sequence coding for the amino acid sequence.

Furthermore, the object according to the invention is achieved by a nucleic acid which codes for an MLP and which comprises the sequence from position 58 to 639 of SEQ ID NO: 1 in which the mutation is at position 67.

[0020]   In a preferred embodiment, the mutation is a T to C mutation.

[0021] Furthermore, the above-mentioned object is MLP and 5 of SEQ ID NO: 1 having a mutation at position 393.
Achieved by a nucleic acid encoding the sequence of positions 8-639.

[0022]   In a preferred embodiment, the mutation is an A and G mutation.

It should be noted that with respect to the position data given above, the latter mentioned relates to the nucleic acid sequence after SEQ ID NO: 1.

Furthermore, the object according to the invention is achieved by a nucleic acid coding for an MLP whose sequence will correspond to a nucleic acid according to the invention without alteration of the genetic code.

Finally, the object according to the invention is achieved by a nucleic acid encoding an MLP, the nucleic acid of which hybridizes to the nucleic acid according to the invention.

In another aspect, the object according to the invention can be achieved by the amino acid sequence encoded by the nucleic acid according to the invention or a part thereof.

[0027] In a preferred embodiment, the amino acid sequence according to the invention has the mutation mentioned above, ie the 10th base of the 2nd exon of the translated sequence or the 11th of the 4th exon of the translated sequence.
It is encoded by the sequence of a nucleic acid according to the invention or a part thereof which contains one or both mutations at position 3 of 2 codons.

In another aspect, the object according to the present invention is achieved by an MLP comprising an amino acid sequence encoded by a nucleic acid according to the present invention or a part thereof, or an MLP comprising an amino acid sequence according to the present invention.

In yet another aspect, the object according to the invention is achieved by a probe for the nucleic acid according to the invention, which probe comprises a sequence according to SEQ ID NO: 4.

In a preferred embodiment said probe is used for the detection and / or amplification of the nucleic acid according to the present invention.

In another aspect, the object according to the invention is achieved by a probe for a nucleic acid according to the invention, the probe of which comprises a sequence corresponding to SEQ ID NO: 5.

In a preferred embodiment, the probe is used for the detection and / or amplification of the nucleic acid according to the present invention.

In another aspect, the nucleic acids according to the invention and / or the probes according to the invention are used separately or in combination for the diagnosis and / or examination of myocardial disease.

[0034]   In this case, in a preferred embodiment, the myocardial disease is dilated cardiomyopathy.

In another aspect, the object according to the invention is achieved by a method for the detection and / or examination of myocardial disease, in which the nucleic acid according to the invention and / or the probe according to the invention is used.

In this case, in a preferred embodiment, the myocardial disease is dilated cardiomyopathy.

In another aspect, the object according to the invention is to digest a sample containing a sequence coding for MLP with a restriction enzyme and to detect the presence of a mutant sequence coding for MLP in ML.
This is achieved by a method for detecting myocardial disease and / or a test method by detecting the occurrence of a restriction enzyme digestion pattern that is relatively changed from the restriction enzyme digestion pattern of the non-mutated sequence encoding P.

In this case, it is provided in the embodiment wherein said myocardial disease is dilated cardiomyopathy.

In another embodiment of the method according to the invention, said method comprises the method according to the invention for the detection and / or examination of myocardial disease, in which the nucleic acid according to the invention and / or the probe according to the invention is used. Is one embodiment of the method according to.

In another embodiment of the method according to the present invention the MLP coding sequence is labeled P
Amplified by CR.

[0041]   In another embodiment, the detection or test is directed to the nucleic acid according to the present invention.

In a particularly preferred embodiment said amplification is carried out with a primer comprising a sequence according to SEQ ID NO: 13 and / or SEQ ID NO: 14.

In a particularly preferred embodiment, the restriction enzyme digestion performed by said Nci I is provided.

In a preferred embodiment, the length of the PCR product is about 308 bp for non-mutated sequences,
The second mutation of the nucleic acid sequence of SEQ ID NO: 1 in which the mutation occurs by the conversion of T and C
In the case of the mutation of the 10th base of the exon or the mutation at the 67th position, about 205 and about 103 bp
Two PCR products with

In the method according to the invention there is provided a detection or test directed to a nucleic acid according to the invention, preferably a nucleic acid having a mutation at position 3 of codon 112 within exon 4 of the translated sequence of SEQ ID NO: 1. sell.

In the method according to the invention, said amplification is SEQ ID NO: 15 and / or SEQ ID NO: 1.
It is provided in one aspect to be carried out with a primer comprising a sequence according to 6.

[0047]   In a preferred embodiment, the restriction enzyme digestion is performed by Cvi RI.

Detection of myocardial disease, in particular dilated cardiomyopathy, using the probe according to the invention, and / or
Alternatively, in the method according to the invention for testing, for the detection or testing of the nucleic acid sequences according to the invention, the hybridization of the sample to be examined is carried out with a probe comprising the sequence according to SEQ ID NO: 4.

In this case, in a preferred embodiment, the hybridization carried out at 60 ° C. is provided.

Detection of myocardial disease, in particular dilated cardiomyopathy, using the probe according to the invention, and / or
Alternatively, in the method according to the invention for testing, for the detection or testing of the nucleic acid sequences according to the invention, the hybridization of the sample to be studied is carried out with a probe comprising the sequence according to SEQ ID NO: 5.

In another aspect, the object of the present invention is achieved by a method for detecting and / or testing myocardial disease by detecting the amino acid sequence according to the present invention or the MLP according to the present invention.

[0052]   In a preferred embodiment, the myocardial disease is dilated cardiomyopathy.

In another preferred embodiment, the amino acid sequence or MLP detected with the antibody
I will provide a.

[0054]   In a preferred embodiment, there is provided an antibody that is a monoclonal antibody.

In another aspect of the invention, said object is provided that the kit comprises at least one nucleic acid according to the invention, and / or at least one probe according to the invention, whereby the nucleic acid according to the invention is The detection kit of

In another aspect, the object according to the invention is that the ML according to the invention comprises at least one antibody against the MLP according to the invention or against the amino acid sequence according to the invention.
This is achieved by P or an amino acid sequence detection kit according to the present invention.

In another aspect, the regulation comprising about 500 base pairs, the object of which is either 500 base pairs upstream from the first base of the first exon of the human genomic sequence encoding MLP. Achieved by nucleic acids.

In an embodiment of a regulatory nucleic acid according to the present invention, a regulatory nucleic acid comprising about 1000 base pairs, of which 1000 base pairs are either upstream from the first base of the first exon of the human genomic sequence encoding MLP. I will provide a.

In another aspect, the object is produced from human genomic DNA by the use of two primers whose upstream primer comprises SEQ ID NO: 9 and downstream primer comprises SEQ ID NO: 10 in a PCR reaction. Achievable regulatory nucleic acid.

In another aspect, said object is achieved by the regulatory sequences including the sequence according to SEQ ID NO: 8.

In one embodiment of the regulatory nucleic acid according to the present invention, a regulatory nucleic acid that is a promoter is provided.

In another aspect, the object according to the invention is achieved by a vector containing the regulatory sequences according to the invention.

In a preferred embodiment, the regulatory sequences contained in the vector pAd CMV ssTnI are provided, in which the CMV promoter has been replaced by the regulatory nucleic acid according to the invention.

In a preferred embodiment, the vector according to the invention is an adenovirus vector.

In another aspect, there is provided a vector of the invention comprising a coding sequence under the control of a regulatory nucleic acid according to the invention.

In yet another aspect, there is provided a coding sequence selected from the group comprising hsp70 and “slow skeletal troponin I”.

In yet another aspect, said object is achieved by a cell comprising a regulatory nucleic acid according to the invention and / or a vector according to the invention.

[0068]   In a preferred embodiment of the cell according to the invention said cell is a eukaryotic cell.

[0069]   In a more preferred embodiment, the cells are mammalian cells.

[0070]   In a highly preferred embodiment, the cells are human cells.

In another aspect, the object according to the invention is achieved by an agent comprising a regulatory nucleic acid according to the invention, a vector according to the invention and / or a cell according to the invention.

[0072]   In a preferred embodiment, the drug is used within the context of gene therapy.

In another preferred embodiment of the agent according to the present invention, an agent for treating and / or preventing myocardial disease is provided.

In a preferred embodiment, point mutations in the gene are indicative of cardiovascular disease resulting in a clinical picture.

In a preferred embodiment of the agent according to the invention, point mutations in the genes encoding the sarcomeric, dystrophin and cardiac actin proteins are provided.

In a very particularly preferred embodiment of the agent according to the invention, there is a cardiovascular disease selected from the group comprising hypertrophic cardiomyopathy, long QT syndrome, and dilated cardiomyopathy.

The present invention is based on the surprising discovery that the genetic cause or sequence involved in the progression of dilated cardiomyopathy in humans is clearly important. This finding is based on the findings of the inventor who found that variants in various exons of the human MLP gene were consistent with dilated cardiomyopathy.

The MLP gene of the human genomic sequence shown here is composed of 6 exons, whereby all exons have a canonical intron-exon transposition, and promoter-specific elements found at the 5'end. To be done. MLP of human genome sequence
The arrangement of the individual exons in the gene can be explained as follows: 1st exon 12983-13011 (28bp total) 2nd exon 22480-22620 (140bp total) 3rd exon 26653-26823 (170bp total) 4th Exons are 28611 to 28746 (total of 135 bp) Exon 5 is 29912 to 30005 (total of 93 bp) Sixth exon is 322113 to 3923 (total of 712 bp) Therefore, the total gene extends to about 20,000 bp.

The first exon in general encodes the 5'untranslated region; the translated region is encoded by the second to sixth exons. Computer-aided promoter analysis is standard TA
It was shown that the TA box was before the first exon.

A large number of patients with familial DCM were analyzed and, in this case, surprisingly, M
It has been discovered that point mutations occur within the coding sequence of the human genomic sequence of the LP gene as well as within the mRNA or cDNA that corresponds to this location.

Patients from the aforementioned family showed a base pair conversion in the second exon (T → C) resulting in a conversion of tryptophan to arginine at amino acid position 4 (W4R). The mutation that causes this conversion is more precisely the translated human
It occurs at the 10th base of the 2nd exon of the genome sequence. The above position corresponds to position 67 of the sequence according to SEQ ID NO: 1. The mutated sequence is referred to below as SEQ ID NO: 2.

The variants in the MLP gene discovered by the inventors give rise to new restriction cleavage planes and can therefore quickly analyze a large patient population.

Starting with this result, a further patient population where the members suffer DCM
The second exon was examined by amplifying it with genomic DNA from a patient suffering from DCM and digesting with the corresponding enzyme. Thus, from the 500 patient population, 6 other patients could be detected with the variant. He then made a family tree and asked the family for a blood sample, if possible. Based on these studies, to date we have found two families in which the disease cosegregates by genotype.

Without becoming a speculator, the following ideas regarding the above variants of the nucleic acid sequences encoding MLPs should be developed.

The wild-type amino acid tryptophan belongs to the heterocyclic amino acids; the arginine that is converted for this purpose, ie present in the mutant nucleic acid, is the strongest basic amino acid. Therefore, this is a structural destruction transformation. The amino acid conversion is not only a highly conserved tryptophan in this part between MLPs of different species (human, porcine, rat, mouse) but also at the amino terminal flanking amino acids of this protein. It resides within a highly conserved region of proteins. This applies not only to the MLP itself, but also to the closest related proteins (CRP1 and CRP2). The amino-terminal region of the protein is required to transport the MLP to its destination within myocytes ("to dock", the sequence is similar in many proteins and is therefore ubiquitous. Existing) can be inferred from this. If this is the case, the modified MLP will not be transported to its destination and will have no biological effect. Computer analysis shows that the 10 amino acids at the amino terminus are M
Only LP, CRP1, and CRP2 were shown to have significant homology. After conversion, W4R no longer showed significant homology to known proteins. Thus, at the amino terminus of the protein, this is uncommon, and in addition, the typical transporter sequence variant W4R does not produce homology to known proteins.

From the above interpretation, the variant in the second exon is associated with DCM in the corresponding patient.
It can be inferred to have a pathologically important function with a certainty of bounding probability in the origin of.

In addition to the above point mutations, we have discovered others related to DCM. In this case, it is a mutation in the 4th exon of the genomic human MLP gene, more specifically a base conversion at the 3rd position of the 112th codon, whereby a conversion between G and A is carried out. This conversion does not coincide with any change in the initial sequence of the MLP gene product thus encoded. The position of the translated sequence corresponding to the position of the genome mentioned above is position 393, given in SEQ ID NO: 1. This mutant sequence is referred to below as SEQ ID NO: 3.

This variant of the MLP gene or its gene product suffers from severe dilated cardiomyopathy,
He found it in a patient who needed to undergo a heart transplant. The cause of the disease was unknown. However, in this patient there is a clearly reduced MLP-mRNA and it is shown that this patient has a homozygous base pair conversion of A and G at position 3 of codon 112 of exon 4. I was able to. This variant thus appears to result in a shortened half-life of mRNA or to mRNA with other properties, eg via modified differential treatment. Therefore, it may be hypothesized that homozygous base pair changes at codon 112 also have pathogenically significant effects.

The nucleic acids disclosed herein are important in many different ways. By contrast of the disclosed mutant MRL sequences with heart disease, especially dilated cardiomyopathy, the study of the aforementioned biological material has revealed whether the sample and hence the individual from which it originated suffers from heart disease or genetic structure. Therefore, it may be carried out to examine whether or not they have at least an inborn risk of suffering from this heart disease. The use of the disclosed nucleic acids can thus be carried out for both prophylactic and diagnostic purposes. In this case, in addition to individual matching studies, the patient population may also be the control for studies that use or rely on the disclosed nucleic acids.

In this case, the sequences claimed herein are not only the sequences according to SEQ ID NO: 2 and SEQ ID NO: 3, but also all other sequences derivable therefrom, and in particular the mutations disclosed herein. Is further shown. In particular, the aforesaid derived sequences are even more susceptible to mutations beyond the two unique mutations disclosed, especially ML.
It is defined below that it also has a sequence encoding P. The mutations mentioned above can be deletions or additions of nucleotides or complete nucleotide sequences, as well as additional point mutations. It is also conceivable that some deletions or additions will be present in the above sequences. These additions and deletions can occur at various points in the sequence. A nucleic acid as disclosed herein comprises a sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 whereby any part of the sequence of the two is separated by a nucleotide or a sequence of several nucleotides. including. An example of the above sequence is the genome M, which has the mutations mentioned above and whose coding regions are separated from each other by introns.
LP sequence.

[0091]   The production of genomic human MLP sequences is described in the Examples herein.

The probes disclosed herein are allele-specific oligonucleotides complementary to the region of the nucleic acid carrying the disclosed mutations. These probes can be used as primers, in particular for amplifying nucleic acids by the polymerase chain reaction and allowing themselves to be detected in small sample volumes. The probe can be used to detect nucleic acids that exhibit mutations either after the polymerase reaction or directly. For this purpose, the probe may be provided in labeled form. These labels may be radioactive or fluorescent markers.

The sequence of the probe or primer for detecting the mutation of the second exon is shown below: AGA TGC CAA ACC GGG GCG, and set forth herein as SEQ ID NO: 4, and the mutation of the fourth exon is The ones to detect are shown below: TTC ACT GCG AAG TTT GGA, and set forth herein as SEQ ID NO: 5.

For the second exon, the sequences of the primers or probes for detecting the wild-type sequence corresponding to each mutation are shown below: AAGA TGC CAA ACT GGG GCG, and as SEQ ID NO: 6 herein. The case described and for the fourth exon is shown below: TTC ACT GCA AAG TTT GGA, and set forth herein as SEQ ID NO: 7.

In the method according to the invention, sample processing steps commonly used for the detection of nucleic acids or gene products and known to the person skilled in the art are carried out. As starting material for example blood or biopsy material may be used.

Where the method according to the invention is aimed at detecting mutations by creating additional cleavage planes for the restriction enzyme which are wild type, ie not present in any of the genomic sequences, mRNA or cDNA, The resulting nucleic acid fragments are typically separated by agarose gel. However, other separation techniques are also conceivable, for example capillary electrophoresis. When using an agarose gel, mutant nucleic acid fragments are visualized, for example by staining with ethidium bromide or as a result of radioactive markers.

When detecting the gene product of one of the nucleic acids described herein by one of the methods according to the invention, any method suitable for the detection of peptides or proteins can be used for this purpose. There is the possibility of using antibodies, especially monoclonal antibodies.

When detecting a peptide or protein encoded by a nucleic acid that exhibits a mutation in the fourth exon that results in a conversion of codon 112 from codon A to G at position 3, this mutation is itself a primary sequence, That is, since it is a silent mutation that is not obvious in terms of amino acid sequence, it is necessary to switch to another detection method that does not focus on the different properties of the gene product due to the above mutation. However, as described above, in this mutation, in particular in homozygous base pair conversion, ie in this mutation possessed by both alleles, in the extent of the gene product, detection of said mutation is carried out by quantification of its mRNA or gene product. In turn it results in a greatly reduced MLP-mRNA.

The kit according to the invention comprises at least one nucleic acid according to the invention, a probe of the nucleic acid mentioned above or a gene product. The kit according to the invention particularly preferably comprises at least one probe specific for one of the two mentioned mutations. In this case, the probe corresponding to wild type may be included as a negative control. The nucleic acid according to the present invention may be included in the kit as a positive and / or negative control. Auxiliary or in addition to the probe, it cleaves at the new face created by the mutation, and restricts the mutated sequence by comparison with the non-mutated sequence and the resulting-truncated-mutated sequence, i.e., restriction length polymorphism. Restriction enzymes from which digestion is obtained may be included in the kit. The nucleic acid, probe or gene product may be provided as a kit in the form of a suitable buffer. The kit can also include a medium according to the invention, which can immobilize nucleic acids, polypeptides or proteins.

[0100]   In another aspect, the invention relates to regulatory nucleic acids, especially promoters.

Hereinafter, the terms regulatory nucleic acid, promoter structure, promoter are used interchangeably.

In a study carried out by the inventor, it was revealed that the sequence located upstream of the first exon of the genomic human MLP sequence with a length of about 2000 bp is very important for muscle-specific expression. Was done. In particular, both TATA, which are important for gene expression
A box and a CAAT box were found upstream of the first exon. Moreover, we have discovered an AP-1 cis-regulatory element known to be important for muscle-specific genes. The described regulatory nucleic acid sequences have the additional consequence that stress can induce MLP expression. Thus, regulatory nucleic acid sequence refers to a tissue-specific promoter structure, which can be further stress induced.

The development of the MLP gene exclusively in muscle tissue (ie striated and smooth muscle) results from the previously mentioned tissue specificity of the regulatory nucleic acid; thus it is a muscle specific promoter.

Gene products or mRNAs that can be determined by a strong promoter can be detected relatively easily by various techniques (eg Northern blot, PCR).
The first exon encodes a 5'untranslated region of only about 30 base pairs. In essence, several TATA box elements that DNA-dependent RNA polymerase II must bind to transcribe a gene are approximately 2 upstream of the first exon.
It is located between 0 and 30 base pairs. Moreover, several transcription initiation sequences are located approximately 10-20 base pairs upstream of the first exon. In addition, some AP-1 sequences are present immediately before the first exon. These sequences bind the so-called "leucine zipper" transcription factor, which is taken up as an important factor especially in stress inducibility of genes. The presence of these sequences supporting the stress inducibility of the MLP gene was discovered by the inventor.

The complete set of AP-1 sequences is found "upstream" within the first 1000 bases. CA
The AT box is found 500 base pairs upstream. These elements are generally found 80-120 base pairs upstream of the gene, but exclude the key factor for this element in the promoters discovered by the present invention. However, it must be noted that it also provides a promoter, mainly a tissue-specific promoter without the CAAT box, but this is not a completely essential element.

In summary, it can be noted that the AP-1 and CAAT sequences are found up to about 1000 base pairs upstream of the first exon. Therefore, about 1 upstream of exon 1
The 000 base pair range seems to be very important for gene expression. In many cases, a stretch of about 500 base pairs (in short starting from the CAAT box) can be shown upstream of the first exon as the nuclear region.

[0107]   The various elements of the regulatory nucleic acids described herein are represented in FIG.

The regulatory sequence according to the invention in another aspect may also be represented by the sequence according to SEQ ID NO: 8.

The production of the regulatory sequences or promoter structures according to the invention can also be carried out by amplifying the corresponding sequences of human genomic DNA with the aid of the polymerase chain reaction with two primers. In doing so, the primer gP1 having the following sequence: ATC TGA TGT GAA GGC TGG was used as the "upstream" primer and is also shown in the sequence listing as SEQ ID NO: 9.

As a “downstream” primer, we used the primer gP2 with the following sequence: CCT GCC TGT GTG GAC TCC and is also shown in the sequence listing as SEQ ID NO: 10.

The regulatory sequences according to the invention can also be present linked to a vector. The vector can be both a plasmid vector and a viral vector. Vectors are typically nucleic acid molecules that are used for nucleic acids, preferably irrelevant, as a container or medium. Generally, the vector has an origin of replication (“origin
)) And a genetic marker which allows the detection of the vector in the host cell.
Moreover, the vector may contain additional elements used to control the translation and / or transcription of the nucleic acid incorporated or contained within the vector.

Both the regulatory sequences according to the invention and the vector according to the invention can be contained intracellularly. Essentially any cell or cell line can be used for this purpose. The cells may be selected from the group consisting of prokaryotic and eukaryotic cells. Within the group of eukaryotic cells, in other words the cells may in particular be selected from the group consisting of yeast cells, insect cells and mammalian cells. Especially in the case of mammalian cells, they may be provided as primary cells, primary cell cultures or established cell cultures.

For various biological systems, including regulatory sequences and the latter, the materials discussed below, as well as the regulatory nucleic acid sequences described herein under the control of the regulatory sequences referred to above, are tissue-specific for nucleic acid sequences, And in particular, a number of viable application of the fact-based enabling of muscle-specific expression is produced.

Many human diseases result from genetic defects. In other words, these clinical features are caused by a monogenic disease, a mutation within one gene. With knowledge of these molecular modifications, the repair of that molecular defect can be carried out by gene therapy.

In the cardiovascular system, several diseases are known in which point mutations within one gene result in a serious clinical picture. These contain mutations in the gene encoding the sarcomeric protein and are associated with hypertrophic cardiomyopathy (Towbin. The Role of
Cytoskeletal Proteins in Cardiomyop
ties. 10, 131-139 (1998)), resulting in long QT syndrome (Gerwer-Langer-Nielsen syndrome, and Romano-Ward syndrome) (
Neyround, N.M. et al. Circ Res 84, 290-29;
7 (1999), as well as dystrophin (Muntni, which causes dilated cardiomyopathy).
F. et al. N Engl J Med 329, 921-5 (1993);
)) Or the cardiac actin gene (Olsen, T. et al .; Science).
280, 750-752 (1998)). These diseases can in principle be treated from the cause by administering the correct gene.

Very recently, various inventors have reported successful adenovirus administration into cardiomyocytes in vitro and in vivo (Hajjar, R. et al.
Proc Natl Acad Sci USA 95, 5251-525;
6 (1998); Donahue, J .; et al. ; Proc Natl A
cad Sci USA 94, 4664-4668 (1997), and Wes
tfall, M .; et al. ; Proc Natl Acad Sci US
A 94, 5444-5449 (1997)). However, the vectors used so far have the drawback that they are controlled by the cytomegalovirus (CMV) promoter. As such, they were expressed not only in myocardium or muscle tissue, but also in any other somatic cell. However, the aforementioned clinical picture is only relevant to cardiomyocytes. The expression of the transformed gene in cells other than cardiomyocytes makes no sense and only increases the risk of gene therapy, ie the occurrence of unwanted mutations, meaning further mutations or immune responses.
The regulatory nucleic acid according to the invention is only expressed in muscle tissue in contrast to the ubiquitously expressed CMV promoter and is therefore very well suited for muscle tissue or muscle cell gene therapy based on its advantages.

In the case of the production of the regulatory nucleic acids according to the invention, and in particular the production carried out with the two primers mentioned above, by a restriction enzyme (eg EcoRI or any other enzyme whose cloning is carried out at its interface) Corresponding sequences that can be detected can generally be added to their ends.

In the production of the vector according to the invention, the procedure is the Westfall et a
l. (Westfall, M. et al .; Proc Natl Acad.
Sci USA 94, 5444-5449 (1997)), it is excised from the shuttle vector pAd CMV ssTnI by the enzyme corresponding to the CMV- promoter, and the shuttle plasmid pAd CMV ssTnI.
In the form of a MLP-promoter (ie a regulatory nucleic acid according to the invention). The plasmid pAd MLP ssTnI produced here was cloned into pJ
Westfall et al. With M17. (Westfall, M. et.
al. Proc Natl Acad Sci USA 94,5444-
5449 (1997)) by the calcium phosphate method.
Adenoviruses with the MLP-promoter, which can be co-transfixed into cells and expressed in a tissue-specific manner, can be obtained by homologous recombination. This virus was expressed as a special case of so-called "slow skeletal troponin I". However, it can be cloned and expressed as any desired gene product by methods known for the MLP-promoter by a suitable vector or shuttle vector. For example, the hsp70 gene responsible for the protective action in myocardium can be placed upstream (Yellon, D. et.
al. J Mol Cell Card 24, 113-124 (1992).
)). In this case, those known to the person skilled in the art and, for example, Maniatis, Frit
sch & Sambrook, Molecular Cloning. Col
Only the standard cloning method described in d Spring Harbor Laboratories, 1989 is required.

In summary, the promoters according to the invention can be cloned for any desired gene product, and therefore the promoters according to the invention can be tissue-specific for any desired viral or vector-based nucleic acid under their control. It is noted that the expression used can be guided. In this regard, the promoters described herein may be used in the case of the diseases mentioned above and described herein, including in particular gene therapy treatment of DCM. In addition to the diseases mentioned explicitly here, all diseases in which tissue-specific expression of nucleic acids, in particular muscle-specific expression, is necessary can also be treated by the use of the regulatory sequences according to the invention.

The invention will be further described on the basis of the following examples, the drawings and the sequence listing, from which the further features and advantages of the invention can be derived.

Example 1. Production of human MLP-cDNA Human MLP-cDNA was produced with the aid of the polymerase chain reaction (= PCR) by using two primers KMLP1 and KMLP2.

[0122]   The sequence of KMLP1 was prepared as SEQ ID NO: 11 and as follows:   GGC AGA CTT GAC CTT GAC CA Indicates.

[0123]   The sequence of KMLP2 was prepared as SEQ ID NO: 12 and as follows:   GAG GTG CGC CGT TTC TCA GA Indicates.

The cDNA obtained as a PCR product by such a method has a length of about 650 bp and contains the entire coding region of mRNA.

2. Production of Human Genome MLP Sequence Starting from the cDNA described in Example 1, the human genome MLP sequence was produced by screening the human genome gene bank using the cDNA. For this purpose, the cDNA used as probe is radiolabeled (Feinb
erg, A .; et al. Anal Biochem 132, 6-13 (1
983)), and hybridized with a gene bank cloned into bacteriophage P1 (Ioannou, PA et al .; Nature).
Genetics 6, 84-89 (1994)). The screening
For example, Maniatis, Fritsch & Sambrook, Molec
ural Cloning, Cold Spring Harbour Lab
It was carried out by use of general methods known to those skilled in the art, as described in oratories, (1989).

The MLP-gene itself is located on chromosome 11p15.1 (Fung, Y
． M. et al. Genomics 28, 602-603 (1995))
.

Next, the clone of the human genome MLP sequence obtained by such a method was sequenced using an automatic DNA sequencer. Positive clones contain a total of 80,000 base pairs and contain the entire human MLP gene composed of a total of 6 exons as already described above.

3. Detection of Mutations in Exons 2 and 4 of the Human Genome MLP Sequence As previously identified, one of the 2 mutations in the MLP sequence discovered by the inventor thereby causes a conversion between T and C. Is the 10th position of the second exon of the human genomic MLP sequence, and the other is the 3rd position of the 112th codon of the 4th exon of the translated sequence which thereby performs the conversion of A and G. is there. Those corresponding to both mutations are mRNA or cDNA obtained therefrom, whereby the mutation position of the 67th base corresponds to mRNA or cDNA in the case of mutation of the second exon, and the mutation position of the fourth exon In some cases, the mutation position of the 393rd base is mRN.
It corresponds to A or cDNA and is also shown in SEQ ID NO: 1.

3.1 Separation of Genomic DNA and Preparation of "Buffy Coat" Whole blood is centrifuged at 3300 g for about 10 minutes at room temperature. After centrifugation, three layers are obtained: the clear upper phase corresponds to plasma, the pale white phase corresponds to white blood cells and the red lower phase corresponds to red blood cells. The white blood cells can now be separated easily by pipette. These are the "buffy coats".

Separation of genomic DNA Separation of genomic DNA was carried out as follows with the aid of the 1997 "Qiagen Blood Kit" and a standard "Eppendorf tabletop centrifuge": a) About 200 μl of buffy coat 1. Add to 5 ml Eppendorf container. 25
μl proteinase K and 200 μl buffer AL were added thereto and vortexed for 15 minutes (mixed with vigorous shaking).

[0131]   b) The mixture was warmed in a 70 ° C. water bath for 10 minutes.

C) 210 μl ethanol (100%) was added to this mixture and again stirred vigorously.

D) A Qiagen column was added to a 2 ml Eppendorf vessel and the mixture of c) was applied to the column. Centrifugation was performed at 6000 g for 1 minute and the column was attached to a new 2 ml Eppendorf vessel. The centrifuged liquid was discarded and the genomic DNA bound to the column.

E) Add 500 μl of Buffer AW to the column and again 6000 g
It was centrifuged for 1 minute. The centrifuged liquid was discarded again and the column was attached to a new 2 ml Eppendorf container.

F) 500 μl of Buffer AW was added back to the column and the maximum speed (
Centrifugation was carried out for 3 minutes at about 15,000 rpm in an Eppendorf tabletop centrifuge. The centrifuged liquid was discarded again.

G) The column was now transferred to a 1.5 ml Eppendorf vessel and 200 μl of pure water preheated to 70 ° C. was added to the column. Incubation was for 1 minute at room temperature and centrifugation was for 1 minute at 6000 g. We have found in the centrifuged liquid, ie the eluate, genomic DNA which is used for other purposes. In another step, the concentration was measured. About 50 mg of genomic DNA was used for the polymerase chain reaction.

Amplification of the 2nd exon of the human genomic MLP sequence For amplification of the 2nd exon, the following sequence corresponding to SEQ ID NO: 13: ACT CTT AGA GAT TGG TTC ACT CC primer GMLP3, and SEQ ID NO: 14 The primer GMLP4 having the following sequence corresponding to: ACC ACA CTA TGA GAA CCA CTG GC was used.

In this case, the following amplification conditions were used: 1.94 ° C. for 4 minutes Then a total of 36 cycles were started with the following conditions: 2.94 ° C. for 45 seconds 3.62 ° C. for 45 seconds 4.72 ° C. 1 min for 36 cycles followed by: 5.72 ° C. for 5 min 3.2 Restriction digestion of the second exon of the human genomic MLP sequence described in 3.1 and a portion of the resulting PCR product Separated on an agarose gel and analyzed to determine its exact size. For the correct size, which is the case in general, another portion was digested with the restriction enzyme NciI at 37 ° C. overnight and separated again the next morning.

PCR for amplification of the second exon yields a PCR product with a size of 308 base pairs. When a base pair conversion between T and C occurs at the 1st position of the 4th codon, the restriction enzyme NciI cuts and produces 2 fragments with sizes of 103 and 205 base pairs.

In the case of homozygotes no PCR product was found with a size of 308 base pairs,
Rather, only 2 fragments are found. In the presence of a heterozygote, ie, a non-mutated for a particular base, ie a healthy allele, and an allele mutated for a particular base, a PCR product with 308 base pairs is further found in the two small fragments. To be done.

The results of this restriction analysis are shown in FIG. 1, whereby FIG. 1 further shows the results of restriction digestion of the second exon of the amplified human genomic MLP sequence that was subjected to agarose gel and isolated. Show clearly. The traces on each of the two outer sides of the agarose gel represent a measure of molecular weight. The trace transcribed by "Wt" shows that wild-type second exon due to no restriction enzyme added to the restriction batch provided. The trace transcribed by "Wt + E" shows the wild type second exon due to the addition of the restriction enzyme NciI to the supplied restriction batch.

Due to the lack of the mutations described herein at the 10th base of the 2nd exon in the human genome wild-type MLP sequence, the interface for NciI is not present in the 2nd exon and is therefore restricted. For the batch, it produces a pattern that is no different from the wild type sequence without restriction enzymes. The trace transcribed by "M" shows the restriction batch made by the variant of the second exon of the human genomic MLP sequence without the restriction enzyme. No additional streaks were found in the gel without the addition of the restriction enzyme and therefore the size of the nucleic acid fragment containing the second exon is not altered by mutation, as is apparent from comparison with the batch referred to as "Wt". . In the case of a restriction batch with a second exon mutated at the 10th base and a restriction enzyme NciI, which results from cleavage of the nucleic acid at the second exon due to the mutagenic design of the interface to said restriction enzyme, and " The trace transcribed by "M + E" results in the design of the two nucleic acid fragments mentioned above, which is also shown in FIG.

By use of this technique, up to now about 500 patients have been examined for the occurrence of the mutations discovered by the present invention. Six patients were found with the mutation.

3.3 Restriction digestion of the 4th exon of the human genomic MLP sequence For the analysis of the 4th exon, the procedure is essentially as in the 2nd exon. However, in this case it has the following sequence corresponding to SEQ ID NO: 15: primer gMLP7.2 with the following: CAA CAA CGC TAT gAg AAA gAC ACC, and the following sequence corresponding to SEQ ID NO: 16: ggA gCT AgA gAg AAT gAC AgC TgC. Primer gMLP8.2 was used under the following PCR conditions: 1.94 ° C. for 4 minutes then start a total of 36 cycles with the following conditions 2.94 ° C. for 45 seconds 3.60 ° C. for 45 seconds 4.72 ° C. 1 minute at 36 cycles followed by: 5.72 ° C. for 5 minutes Some of the PCR products obtained were separated by agarose gel and analyzed to determine the correct size. For the correct size, which is the general case, another portion was digested with the restriction enzyme Cvi RI at 37 ° C. overnight and separated again on the agarose gel the next morning. It should be noted that for said incubation, the limiting batch of incubation could also be carried out at room temperature for 30-60 minutes. The overnight incubation was performed just in case.

3.4 Detection of mutations in exon 2 or exon 4 using allele-specific oligonucleotides In addition to performing restriction analysis, probes (so-called allele-specific oligonucleotides, ASO) were added to genomic humans. It can also be produced by finding variants in the second and fourth exons of the MLP sequence or the corresponding cDNA or by the mutations described herein.

For the detection of variants or mutations in the second exon, in this case SEQ ID NO: 4
A probe having the following sequence corresponding to: AGA TGC CAA AC C GGG GCG may be used.

[0147] In order to detect the second exon of the wild-type form, the following sequence corresponding to SEQ ID NO: 6: can be used a probe with AGA TGC CAA AC T GGG GCG.

The melting temperature during detection of mutations by using the sequence according to SEQ ID NO: 4 is 60
C. and the melting temperature during detection of wild type by use of the sequence according to SEQ ID NO: 6 is about 58.degree.

For the detection of variants or mutations in the fourth exon, in this case SEQ ID NO: 5
A probe having the following sequence corresponding to: TTC ACT GC G AAG TTT GGA may be used.

For detection of the wild-type form of the fourth exon, a probe having the following sequence, which corresponds to SEQ ID NO: 7: TTC ACT GC A AAG TTT GGA, may be used.

The melting temperature during the detection of mutations by using the sequence according to SEQ ID NO: 5 is 52
C. and the melting temperature during detection of wild type by use of the sequence according to SEQ ID NO: 7 is about 50.degree.

A second primer identified by restriction and probe analysis with the indicated primers.
All vehicle mutations of exons and exons 4 are also sequenced using standard techniques. In all cases, the sequence analysis supported the results of restriction analysis and probe analysis. Thus, especially by use of the probes described herein,
Sequencing of the second or fourth exon, or the corresponding cDNA, or the corresponding cDNA of the genomic human MLP gene or the genomic MLP gene, is used to detect myocardial disease and / or test for myocardial disease, especially dilated cardiomyopathy Can also be used as in the method according to the invention.

3.5 Regulatory Nucleic Acid (Sequence) Structure The regulatory nucleic acid set forth in SEQ ID NO: 8 is shown in FIG. 2 along with labeled promoter-specific elements.

Separation of this regulatory nucleic acid is possible by using the abovementioned primers gP1 and gP2 corresponding to SEQ ID NO: 9 and SEQ ID NO: 10. By using the above-mentioned two kinds of primers, a regulatory nucleic acid sequence or an MLP promoter corresponding in this respect (1000 bp)
Can be amplified.

[0155]   With respect to FIG. 2, the following may be noted herein:   The last sequence underlined at the 3'end is the first exon.

The AP-1 sequence due to the recognition of at most 2 incorrect sets from the consensus sequence TGAG / CTCA is labeled by underlining.

In this regard, it should be noted that the AP-1 and CAAT sequences overlap at the beginning of the promoter, whereby the CAAT sequences are underlined.

Label the TATA box in bold and underlined. The entire disclosures of the various references cited herein are incorporated by reference. The features of the invention disclosed in the description, the claims and the drawings can be essential both for the implementation of the invention in its various aspects, both individually and in any combination.

[Brief description of drawings]

[Figure 1]   3 shows restriction analysis of the second exon of the genomic human MLP gene.

[Fig. 2]   1 shows the sequence of a regulatory nucleic acid according to the invention with promoter-specific elements.

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 9/00 A61P 43/00 105 4H045 9/04 C07K 14/47 43/00 105 C12N 1/15 C07K 14 / 47 1/19 C12N 1/15 C12Q 1/48 Z 1/19 1/68 A 5/10 Z C12Q 1/48 C12N 15/00 ZNAA 1/68 5/00 A A61K 37/02 (81) Designated Country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, Z ), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW F terms (reference) 4B024 AA01 AA11 CA04 CA09 DA03 EA02 FA02 HA14 HA17 4B063 QA19 QQ03 QQ48 QR08 QR14 QR55 QR62 QS25 QS34 4B065 AA90 AA93 AB01 BA02 CA44 4C084 AA02 A A03 AA07 AA13 BA35 CA01 CA25 NA14 ZA362 ZA372 ZB212 4C087 AA01 AA02 BB65 BC83 CA12 NA14 ZA36 ZA37 ZB21 ZC41 4H045 AA10 AA30 CA40 EA23 EA50 HA05

Claims (48)

[Claims] 1. A tenth exon of the second exon of the translated sequence encoding MLP.
A nucleic acid comprising the sequence of SEQ ID NO: 1 having a mutation in the base. 2. The nucleic acid according to claim 1, wherein the mutation is exchange of T and C. 3. The 11th of the 4th exon of the translated sequence encoding MLP.
A nucleic acid comprising the sequence of SEQ ID NO: 1 having a mutation at the 3rd position of 2 codons. 4. The nucleic acid according to claim 3, wherein the mutation is exchange of A and G. 5. The nucleic acid according to any one of claims 1 to 4, which comprises only a sequence encoding an amino acid sequence. 6. A nucleic acid comprising a sequence from positions 58 to 639 of SEQ ID NO: 1, which encodes MLP and in which the mutation is at position 67, in particular a T to C mutation. 7. An MLP encoding and wherein the mutation is at position 393,
A nucleic acid comprising a sequence from positions 58 to 639 of SEQ ID NO: 1, which is particularly an A to G mutation. 8. A nucleic acid encoding an MLP, the sequence of which corresponds to the nucleic acid according to any one of claims 1 to 7 without the degeneracy of the genetic code. 9. A nucleic acid encoding an MLP, wherein said nucleic acid hybridizes with the nucleic acid according to any one of claims 1-8. 10. The nucleic acid according to any one of claims 1 to 9 or a part thereof,
In particular, an amino acid sequence encoded by the above sequence or a part thereof carrying a mutation. 11. An MLP comprising the amino acid sequence encoded by the nucleic acid according to any one of claims 1 to 9 or a part thereof, or the amino acid sequence according to claim 10. 12. A probe comprising the sequence SEQ ID NO: 4 for the nucleic acids according to any one of claims 1-9, especially for their detection or amplification. 13. A probe for the nucleic acid according to any one of claims 1 to 9, which probe comprises the sequence SEQ ID NO: 5, especially for their detection or amplification. 14. Use of the nucleic acid according to any one of claims 1 to 9 or the probe according to claim 12 or 13 for the diagnosis and / or examination of cardiomyopathy, in particular dilated cardiomyopathy. 15. A method for detecting and / or testing myocardial disease, in particular dilated cardiomyopathy, wherein the nucleic acid according to any one of claims 1 to 9 and / or the method according to claim 12 or 13. The above method using a probe. 16. A method for detecting and / or testing myocardial disease, in particular dilated cardiomyopathy, according to claim 15, wherein a probe containing a sequence encoding MLP is digested with a restriction enzyme, and MLP is used. The method as described above, wherein the presence of a mutated sequence coding for MLP. 17. The method of claim 16, wherein the MLP coding sequence is amplified using PCR prior to digestion. 18. The method according to claim 16 or 17, wherein the detection or test of nucleic acid is directed to the nucleic acid according to any one of claims 1-9. 19. The method according to claim 17 or 18, wherein the amplification is carried out with a primer comprising a sequence according to SEQ ID NO 13 and / or SEQ ID NO 14. 20. The method of any one of claims 16-19, wherein the restriction enzyme digestion is performed with Nci I. 21. In the case of a non-mutated sequence, the PCR product is about 308 bp in length, and the second mutation of the nucleic acid of SEQ ID NO: 1 results from the conversion of T and C.
21. The method of claim 20, wherein the PCR product of 2 has a length of about 205 and about 103 bp in the case of a mutation at base 10 or position 67 of the exon. 22. The method according to any one of claims 15 to 17, wherein the detection or test of the nucleic acid is directed to the nucleic acid according to any one of claims 3 to 9. 23. The method of claim 22, wherein the amplification is performed with a primer comprising the sequence set forth in SEQ ID NO: 15 and / or SEQ ID NO: 16. 24. The method according to claim 22 or 23, wherein the restriction enzyme digestion is performed by Cvi RI. 25. To detect or test the nucleic acid sequence according to any one of claims 1 to 9, hybridization of a sample to be investigated with a probe comprising the sequence according to SEQ ID NO: 4 is carried out. The method according to claim 15. 26. The method of claim 25, wherein the hybridization is performed at 60 ° C. 27. The method according to claim 15, wherein the hybridization of the sample to be investigated with a probe comprising the sequence set forth in SEQ ID NO: 5 is carried out for the detection or examination of nucleic acid sequences. 28. A method for detecting and / or testing a myocardial disease, in particular dilated cardiomyopathy, wherein the amino acid sequence according to claim 10 is detected or.
The method as described above, wherein the MLP is detected. 29. The method according to claim 28, wherein the amino acid sequence or MLP is detected using an antibody, preferably a monoclonal antibody. 30. A kit for detecting the nucleic acid according to any one of claims 1 to 9, wherein at least one nucleic acid according to any one of claims 1 to 9 and / or 12 Or the above kit comprising at least one probe according to 13; 31. The MLP according to claim 11 or at least one antibody against the amino acid sequence according to claim 10, wherein the MLP according to claim 11 or the amino acid sequence encoding the MLP according to claim 10. Detection kit. 32. A regulatory nucleic acid comprising about 500 base pairs upstream from the first base of the first exon of the human genomic sequence encoding MLP. 33. The regulatory nucleic acid of claim 32, which comprises about 1000 base pairs upstream from the first base of the first exon of the human genomic sequence encoding MLP. 34. A regulatory nucleic acid, which can be produced from human genomic DNA by PCR reaction by using two primers, an upstream primer containing SEQ ID NO: 9 and a downstream primer containing SEQ ID NO: 10. 35. A regulatory nucleic acid comprising the sequence set forth in SEQ ID NO: 8. 36. The regulatory nucleic acid according to any one of claims 32-35, wherein the regulatory nucleic acid is a promoter. 37. A vector comprising the regulatory sequence according to any one of claims 32 to 36. 38. The CMV promoter within the vector according to claim 32 to 36.
The regulatory sequence is replaced with the vector p by substituting the regulatory sequence according to any one of 1.
38. The vector of claim 37 contained within Ad CMV ssTnI. 39. The vector according to claim 37, which is an adenovirus vector. 40. A vector according to any one of claims 37 to 39 comprising a coding sequence under the control of the regulatory nucleic acid according to any one of claims 32 to 36. 41. The vector of claim 40, wherein said coding sequence is selected from the group comprising hsp70 and “slow skeleton troponin I”. 42. A cell comprising the regulatory nucleic acid according to any one of claims 32 to 36 and / or the vector according to any one of claims 37 to 40. 43. The cell according to claim 42, wherein the cell is a eukaryotic cell, preferably a mammalian cell, more preferably a human cell. 44. A drug comprising the regulatory nucleic acid according to any one of claims 32 to 36, the vector according to any one of claims 37 to 41 or the cell according to claim 42 or 43. 45. The drug according to claim 44, wherein the drug is used within the framework of gene therapy. 46. The agent according to claim 44 or 45, wherein the agent is for treating and / or preventing cardiovascular disease, particularly cardiovascular disease in which a point mutation in a gene causes a clinical picture. 47. The agent according to claim 46, wherein the point mutation is present in genes encoding proteins of sarcomere, dystrophin, and cardiac actin. 48. The drug according to claim 46 or 47, wherein the cardiovascular disease is selected from the group including hypertrophic cardiomyopathy, long QT syndrome, and dilated cardiomyopathy.