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WO2012107580A1 - Procédé de diagnostic in vitro pour prédire une prédisposition à la cardiomyopathie - Google Patents

Procédé de diagnostic in vitro pour prédire une prédisposition à la cardiomyopathie Download PDF

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Publication number
WO2012107580A1
WO2012107580A1 PCT/EP2012/052352 EP2012052352W WO2012107580A1 WO 2012107580 A1 WO2012107580 A1 WO 2012107580A1 EP 2012052352 W EP2012052352 W EP 2012052352W WO 2012107580 A1 WO2012107580 A1 WO 2012107580A1
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bag3
hspb7
cardiomyopathy
dcm
gene
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Philippe Charron
Eric Villard
François CAMBIEN
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Institut National de la Sante et de la Recherche Medicale INSERM
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Institut National de la Sante et de la Recherche Medicale INSERM
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure

Definitions

  • the invention relates to the field of molecular diagnosis.
  • the invention provides in vitro diagnosis methods for detecting a genetic predisposition to cardiomyopathy, such as dilated cardiomyopathy. More specifically, the invention relates to an in vitro diagnosis method for detecting a genetic predisposition to cardiomyopathy in a human subject, said method comprising the steps of: a. providing a biological sample from a human subject; and, b. detecting the presence or absence of an HSPB7 disease associated variant and/or BAG3 disease associated variant in said biological sample, wherein the presence of said HSPB7 and/or BAG3 disease associated variant is indicative of a genetic predisposition for cardiomyopathy in said human subject.
  • BACKGROUND OF THE INVENTION BACKGROUND OF THE INVENTION:
  • HF Heart failure
  • Idiopathic dilated cardiomyopathy is a form of HF defined by the presence of left ventricular dilatation and left ventricular systolic dysfunction in the absence of an obvious etiology such as coronary artery disease (CAD), hypertension, valvular disease, or congenital defect 3 ' 4 ' 5 .
  • DCM is a major cause of systolic heart failure and the leading indication for heart transplantation 6 , it affects approximately 1/2500 adults and it is more common in men than in women 7 .
  • DCM The pathophysiology of DCM is poorly understood 3 .
  • the disease is considered to be multifactorial with a possible implication of environmental factors and the existence of a strong genetic component attested by a high rate of familial aggregation 3 ; 20% to 35% of DCM cases having an affected first-degree relative 3 ' 4 .
  • Genetic analyses of monogenic DCM have identified mutations in more than 30 genes, most of them encoding proteins of the cytoskeleton or the sarcomere 8 . These genes may carry mutations that are implicated in familial forms of the disease cases as well as common susceptibility alleles that are over- represented in sporadic cases 9 .
  • the genetic basis of DCM whether familial or sporadic, is still largely unresolved and results of candidate gene association studies have been inconsistent 10 .
  • DCM dilated cardiomyopathy
  • discovery and replication cohorts comprising overall 2344 cases and 2410 controls
  • the inventors identified DCM biomarkers and in particular two DCM-associated S Ps, rsl0927875 and rs2234962 with respective -values of 9.5 x 10 "10 and 4.0 x 10 "12 in the combined data set.
  • the first SNP is located at a locus on lp36.13 which exhibit a yin/yang haplotype structure encompassing several genes including HSPB7.
  • the second SNP on 10q26.11 is located within BAG3 and is non-synonymous.
  • the inventors identified several damaging mutations which were absent in healthy individuals, suggesting that they are causal for DCM.
  • a first object of the invention relates to an in vitro diagnosis method for detecting a genetic predisposition to cardiomyopathy in a subject, said method comprising the steps of: a. providing a biological sample from said subject; and, b. detecting the presence or absence of an HSPB7 disease associated variant
  • HSPB7 and/or BAG3 disease associated variant in said biological sample, wherein the presence of said HSPB7 and/or BAG3 disease associated variant is indicative of a predisposition to cardiomyopathy in said subject.
  • the method is particularly useful in predicting a predisposition to dilated cardiomyopathy, including familial or idiopathic dilated cardiomyopathy.
  • said HSPB7 disease associated variant is a genetic mutation that decreases the expression level of HSPB7 gene without resulting in an alteration of the predicted amino acid sequence encoded by the HSPB7 gene.
  • Said genetic mutation may be for example, one or more single nucleotide polymorphism (SNP) mutations in HSPB7 gene locus.
  • SNP single nucleotide polymorphism
  • said genetic mutation is a SNP rsl0927875 and/or rs945417.
  • said BAG3 disease associated variant is a genetic mutation altering the predicted amino acid sequence encoded by the BAG3 gene.
  • said genetic mutation includes a non-synonymous single nucleotide polymorphism (SNP) in a BAG3 coding region.
  • said genetic mutation is a genetic mutation resulting in a deletion or a dysfunction of the BAG domain of the Bag3 protein.
  • said genetic mutation is a non-synonymous single nucleotide polymorphisme in the BAG domain of the Bag3 protein.
  • said non-synonymous SNP in BAG3 coding region is rs2234962 or rs3858340.
  • the invention naturally further relates to a kit for carrying out the above-described method, said kit comprising: a. means for detecting one or more SNPs in BAG3 and/or HSPB7 gene loci, and b. optionally, instructions for use of the kit.
  • the invention also relates to a kit for carrying out the above-described method, said kit comprising: a. means for detecting causal mutations in the full gene sequence of BAG3; and, b. optionally, instructions for use of the kit.
  • the invention relates to an in vitro diagnosis method for detecting a genetic predisposition to cardiomyopathy in a subject, said method comprising the steps of: a. providing a biological sample from said subject; and, b. detecting the presence or absence of an HSPB7 disease associated variant and/or BAG3 disease associated variant in said biological sample, wherein the presence of said HSPB7 and/or BAG3 disease associated variant is indicative of a genetic predisposition to cardiomyopathy in said subject.
  • a subject has a genetic predisposition to a disease when this subject has a higher risk to develop such disease, compared to the average risk in a population to develop such disease.
  • a predisposition does not mean that the subject will develop the disease.
  • "Detecting a predisposition to cardiomyopathy” therefore includes detecting a higher risk of developing the disease, or determining the susceptibility of that subject to developing the disease or to having a poor prognosis for the disease.
  • cardiomyopathy refers to all myocardial disorder in which the heart muscle is structurally and functionally abnormal, in the absence of coronary artery disease, hypertension, valvular disease and congenital heart disease sufficient to cause the observed myocardial abnormality (Eur Heart J 2008;29:270-276).
  • Cardiomyopathy includes without limitation, hypertrophic cardiomyopathy (HCM or HOCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), isolated ventricular non- compaction, mitochondrial myopathy, dilated cardiomyopathy such as familial or idiopathic dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), Takotsubo cardiomyopathy and Loeffler endocarditis.
  • HCM hypertrophic cardiomyopathy
  • ARVC arrhythmogenic right ventricular cardiomyopathy
  • isolated ventricular non- compaction mitochondrial myopathy
  • dilated cardiomyopathy such as familial or idiopathic dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), Takotsubo
  • the diagnosis methods may also be applied to other cause of heart failure such as systolic or diastolic left ventricular dysfunction due to coronary artery disease, valvular diseases or arterial.
  • the invention more specifically relates to a method for detecting a genetic predisposition to dilated cardiomyopathy including familial or idiopathic dilated cardiomyopathy.
  • the method of the invention can be carried out on any appropriate biological sample obtained from a subject.
  • biological sample refers to a sample that contains either nucleic acid or protein materials reflecting the genomic information of cells, tissue or organs of the subject.
  • said sample is obtained from a mammal, for example from rodents, cats, dogs, horses, primates or human.
  • said sample is obtained from a human subject.
  • said biological sample may be obtained from urine, blood including without limitation peripheral blood or plasma, stool, sputum, bronchoalveolar fluid, endotracheal aspirates, wounds, cerebrospinal fluid, lymph node, exsudate and more generally any human biopsy tissue or body fluids, tissues or materials.
  • said biological sample is blood, more preferably human blood sample.
  • a "disease associated variant” means any genotypic biomarker, such as a genetic mutation, that is associated with an increased or decreased risk of developing the disease.
  • said disease associated variant is a genetic mutation in HPSB7 or BAG3 gene locus.
  • a “genetic mutation” refers to a nucleotide change (or nucleotide changes) in the wild type sequence of the corresponding gene.
  • Said genetic mutation refers to a germline mutation that can be considered as a causal mutation (present only in patients, usually in a familial form of the disease, with a direct causal link with the disease).
  • mutations can occur within a gene or chromosome, including specific changes in non-coding regions of a chromosome, for instance changes in or near regulatory regions of genes.
  • Types of mutations include, but are not limited to, base substitution point mutations (which are either transitions or transversions), deletions, and insertions.
  • Missense mutations are those that introduce a different amino acid into the sequence of the encoded protein; nonsense mutations are those that introduce a new stop codon; and silent mutations are those that introduce the same amino acid often with a base change in the codon.
  • mutations can be in-frame (not changing the frame of the overall sequence) or frame shift mutations, which may result in the misreading of a large number of codons (and often leads to abnormal termination of the encoded product due to the presence of a stop codon in the alternative frame).
  • a disease associated variant is a single nucleotide polymorphism (S P) in HSPB7 or BAG3 gene locus.
  • a “single nucleotide polymorphism (S P)" is a single base (nucleotide) polymorphism in a DNA sequence among individuals in a population.
  • a single nucleotide polymorphism (SNP) may fall within coding sequences of genes, non-coding regions of genes, or in the intergenic regions between genes. SNPs within a coding sequence will not necessarily change the amino acid sequence of the protein that is produced, due to degeneracy of the genetic code.
  • a SNP in which both forms lead to the same polypeptide sequence is termed “synonymous” (sometimes called a silent mutation)-if a different polypeptide sequence is produced they are “nonsynonymous”.
  • a nonsynonymous change may either be missense or "nonsense", where a missense change results in a different amino acid, while a nonsense change results in a premature stop codon.
  • the exact sequence of a SNP can be determined from the database of SNPs available at the NCBI website (Entrez SNP, dbSNP build 128, Jan. 28, 2009).
  • the "position" of the nucleotide of interest gives the location in the genome of the SNP, referring to the nucleotide position from the p-terminus of the chromosome in the human genome, see the NCBI SNP website (dbSNP), available on the internet.
  • the inventors have identified specific single nucleotide polymorphisms of HSPB7 that are associated to a higher risk of developing dilated cardiomyopathy.
  • the inventors have further shown that such SNPs may be associated to a decreased level of expression of HSPB7 gene compared to expression level with a wild type sequence.
  • predisposition to cardiomyopathy may be diagnosed by detecting a genetic mutation in HSPB7 gene locus that decreases the expression level of HSPB7 gene in a biological sample obtained from a subject.
  • human wild type HSPB7 gene is the gene comprising the nucleotide sequence as shown in Genbank accession number (NM_014424.4 GI: 164519093 :
  • said genetic mutation is a single nucleotide polymorphism (SNP) mutation in HSPB7 gene locus, which SNP in HSPB7 gene locus is associated with a decreased expression level of HSPB7 gene.
  • SNP single nucleotide polymorphism
  • HSPB7 gene locus may comprise in the context of the present invention, the genetic sequence of HSPB7, the region of the promoter, the introns, the exons including 3' and 5' untranscribed regions, and some intergenic regions involved in HSPB7 gene expression.
  • the genetically associated region discovered by the inventors is covering 5 genes in the locus and extended from SPEN to CLC KB (on chromosome Ip36.2-p36.1 interval 162670000-163700000; GRCh37/hgl9 assembly) which comprise several other genes: SPEN (spen homolog, transcriptional regulator), HSPB7 (heat shock 27 kDa protein family, member 7), CLCNKA (chloride channel Ka) and CLCNKB (chloride channel kb).
  • said genetic mutation is the SNP rs 10927875 or rs945417 as described for example in dbSNP
  • rsl0927875 is a SNP located in an intron of ZBTB17 (also referred as MIZ-1) (position 16299312; genomic release GRCh37).
  • Rs945417 is a SNP located at position 16344625 (genomic release GRCh37) and located in the proximal promoter region of HSPB7 gene sequence.
  • SNPs in HSPB7 gene locus may be detected in addition to rsl0927875 and/or rs945417, in particular, other SNPs which are associated with a decreased expression of HSPB7.
  • SNPs in HSPB7 have been described for example in Matkovitch et al. 21 , to be associated to systolic heart failure.
  • the inventors have further identified specific SNPs of BAG3 gene that are associated to a higher risk of developing dilated cardiomyopathy.
  • SNPs includes non-synonymous mutations in the coding regions and have been shown to be associated to an increased risk of developing cardiomyopathy. In some cases, the increased risk may therefore be linked to an altered Bag3 protein sequence. Sequencing of BAG3 exons in patients with familial dilated cardiomyopathy identified several damaging mutations absent in healthy individuals, suggesting a causal link of the mutations for dilated cardiomyopathy.
  • said genetic mutation is one or more single nucleotide polymorphism (SNP) mutations in BAG3 gene locus, for example, non synonymous single nucleotide polymorphism in BAG3 coding regions.
  • SNP single nucleotide polymorphism
  • genetic predisposition to cardiomyopathy may be diagnosed by detecting a genetic mutation that alters the predicted amino acid sequence encoded by the BAG3 gene in the biological sample.
  • BAG3 gene locus may comprise in the context of the present invention, the genetic sequence of BAG3, the region of the promoter, the introns, the exons including 3' and 5' untranscribed regions, and some intergenic regions involved in HSPB7 gene expression.
  • the BAG3 gene is located on chromosome lOq (position chrlO: 121400000- 121440000; GRCh37/hgl9 assembly).
  • human wild type BAG3 gene is the gene comprising the nucleotide sequence as shown in Genbank accession number (NM_004281.3 GL62530382; with corresponding cDNA of SEQ ID NO:3), and coding for the polypeptide sequence of SEQ ID NO:4 (NP_004272.2 GI: 14043024).
  • said genetic mutation is the single nucleotide polymorphism rs2234962 or rs3858340 as described for example in dbSNP
  • the SNP rs2234962 is a non-synonymous SNP (c. T757C, p. C151R) located within the coding sequence of BAG3 on chromosome 10q25.2-q26.2 (position 121429633 of GRCh37 Assembly).
  • the SNP rs3858340 is a non-synonymous SNP (c. C1526T, p. P407L) located within the coding sequence of BAG3 on chromosome 10q25.2-q26.2 (position 121436286 of GRCh37 Assembly).
  • BAG3 gene locus may be detected in addition to rs2234962 or rs3858340.
  • said genetic mutation is one or more of the following SNPs set forth in Table 1, which have been identified in familial forms of dilated cardiomyopathy.
  • SNPs in BAG3 coding regions set forth in Table 1 may be detected in the diagnosis methods according to the invention, either as disease associated variants or in combination with detecting SNPs rs2234962 or rs3858340: Table 1:
  • said BAG3 disease associated variant is a variant that is not associated to muscular dystrophy.
  • said BAG3 disease associated variant is a Bag3 mutation resulting in a deletion or a dysfunction in the BAG domain of the Bag3 protein, for example one or more amino acid substitution or deletion in the BAG domain of Bag3 protein.
  • BAG domain may be defined by the following amino acid sequence of SEQ ID NO:75, corresponding to residues at amino acid position 421 to 498 of the Bag3 amino acid sequence of SEQ ID NO:4.
  • the above-mentioned disease associated variants for HSPB7 or BAG3 may be detected in the biological sample either at the nucleic acid level or at the polypeptide level and includes methods to detect a genetic mutation in the genomic DNA or RNA transcripts and method to detect abnormal expression of the gene product or mutation in the gene product (RNA transcripts or polypeptide).
  • the presence of the genetic mutation may be detected on either one or both chromosomes, wherein the identification of the genetic mutation (e.g. one or more specific SNPs as described above) on at least one chromosome indicates that the subject is at risk for developing cardiomyopathy, the subject has a cardiomyopathy, or has an early stage of cardiomyopathy.
  • the identification of the genetic mutation e.g. one or more specific SNPs as described above
  • a variety of techniques are known in the art for detecting a genetic mutation within a sample, including sequencing genotyping, microarrays, Restriction Fragment Length Polymorphism, Southern Blots, SSCP, dHPLC, single nucleotide primer extension, allele- specific hybridization, allele-specific primer extension, oligonucleotide ligation assay, and invasive signal amplification, MALDI-TOF mass spectrometry and fluorescence polarization (FP).
  • sequencing genotyping microarrays, Restriction Fragment Length Polymorphism
  • Southern Blots Southern Blots
  • SSCP Southern Blots
  • dHPLC single nucleotide primer extension
  • allele-specific hybridization allele-specific primer extension
  • oligonucleotide ligation assay oligonucleotide ligation assay
  • invasive signal amplification MALDI-TOF mass spectrometry and fluorescence polarization (FP).
  • detecting a genetic mutation may be carried out by sequencing a nucleic acid comprising a fragment of BAG3 or HSPB7 gene locus and analysing the sequence for detecting the presence or the absence of said genetic mutation.
  • PCR amplification may be performed on genomic DNA from said biological sample allowing amplification of the fragments to be sequenced.
  • primers which span one or more fragments that comprise the putative location of a BAG3 or HSPB7 genetic mutation may be used to detect, by sequencing, said BAG3 or HSPB7 genetic mutations.
  • primers which may be used in the diagnosis methods of the invention are short nucleic acid molecules, for instance DNA oligonucleotides of 10 nucleotides or more in length, which can be annealed to the complementary target nucleic acid molecule by nucleic acid hybridization to form a hybrid between the primer and the target nucleic acid strand.
  • a primer can be extended along the target nucleic acid molecule by a polymerase enzyme. Therefore, primers can be used to amplify the target nucleic acid molecule, such as fragments of BAG3 coding regions or HSPB7 gene locus. The specificity of a primer increases with its length.
  • a primer that includes 30 consecutive nucleotides will anneal to a target sequence with a higher specificity than a corresponding primer of only 15 nucleotides.
  • probes and primers can be selected that include at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more consecutive nucleotides.
  • a primer is at least 15 nucleotides in length, such as at least 15 contiguous nucleotides complementary to a target nucleic acid molecule.
  • primers having at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or more contiguous nucleotides complementary to the target nucleic acid molecule to be amplified, such as a primer of 15-70 nucleotides, 15-60 nucleotides, 15-50 nucleotides, or 15-30 nucleotides.
  • An “upstream” or “forward” primer is a primer 5' to a reference point on a nucleic acid sequence.
  • a “downstream” or “reverse” primer is a primer 3' to a reference point on a nucleic acid sequence.
  • at least one forward and one reverse primer are included in an amplification reaction.
  • Nucleic acid probes and primers can be readily prepared based on the nucleic acid sequence of HSPB7 or BAG3 gene locus, for example SEQ ID NO: l and SEQ ID NO:3.
  • PCR primer pairs can be derived from a known sequence by using computer programs intended for that purpose such as Primer 3 (v. 0.4.0 Whitehead Institute for Biomedical Research, Steve Rozen, and Helen Skaletsky).
  • primers that may be used for amplifying BAG3 and/or HSPB7 exons prior to sequencing are shown in the following table 2.
  • Typical PCR amplification conditions that may be used are: 95°C 5 min followed by 35 cycles :[95°C-15sec / Tm (as shown in Table2) - 30sec / Elongation Temp - 30sec].
  • Table 2 Examples of PCR primers and conditions for sequence of BAG3 and HSPB7
  • BAG3 exon primers (5'>3') 5' position (ref hgl9) PCR: size;Tm;MgC12;DMSO
  • the genetic mutations are detected by specific hybridization of nucleic acid probes, such as oligonucleotide probes to genomic DNA or RNA transcripts or corresponding cDNA, containing the BAG3 or HSPB7 genetic mutations, for example containing the specific BAG3 or HSPB7 SNPs as described in the above paragraphs.
  • nucleic acid probes such as oligonucleotide probes to genomic DNA or RNA transcripts or corresponding cDNA
  • BAG3 or HSPB7 genetic mutations for example containing the specific BAG3 or HSPB7 SNPs as described in the above paragraphs.
  • Taqman 5' nuclease genotyping method is used as described in the Examples.
  • oligonucleotide PCR primers are designed that flank the mutation in question and allow PCR amplification of the region.
  • a third oligonucleotide probe is then designed to hybridize to the region containing the base subject to change between different alleles of the gene.
  • This probe is labelled with fluorescent dyes at both the 5' and 3' ends. These dyes are chosen such that while in this proximity to each other the fluorescence of one of them is quenched by the other and cannot be detected.
  • Extension by Taq DNA polymerase from the PCR primer positioned 5' on the template relative to the probe leads to the cleavage of the dye attached to the 5' end of the annealed probe through the 5' nuclease activity of the Taq DNA polymerase. This removes the quenching effect allowing detection of the fluorescence from the dye at the 3' end of the probe.
  • the discrimination between different DNA sequences arises through the fact that if the hybridization of the probe to the template molecule is not complete (there is a mismatch of some form) the cleavage of the dye does not take place. Thus only if the nucleotide sequence of the oligonucleotide probe is completely complimentary to the template molecule to which it is bound will quenching be removed.
  • a reaction mix can contain two different probe sequences each designed against different alleles that might be present thus allowing the detection of both alleles in one reaction.
  • primers and probes useful for detecting the SNPs of BAG and/or HSPB7 gene associated to dilated cardiomyopathy are shown in the following Table 3 : Table 3: Primers (a) and probes (b) and reaction conditions for Taqman genotyping
  • the method of detecting a genetic mutation in BAG3 or HSPB7 gene locus comprises use of a restriction enzyme that specifically recognizes sequence corresponding to wild type or genetic mutations of BAG3 or HSPB7 gene sequence.
  • said disease associated variant may be detected by detecting abnormal expression of gene product such as mRNA transcripts.
  • Methods to quantify mRNA level from total mRNA cell extract are well known in the art and include quantitative PCR such as Real Time PCR amplification with labelled probes.
  • Abnormal expression level may be determined by comparing the expression level to a control level. Significant difference in the expression level as determined by statistical analysis is indicative of abnormal expression level.
  • a polypeptide disease associated variant is detected using a binding agent that specifically binds to a polypeptide disease associated variant.
  • a binding agent that specifically binds to a mutant variant of Bag3 corresponding to said disease associated variant but not to wild type Bag3 may be used.
  • Said binding agent may be for example an antibody or antibody fragments comprising antigen-binding regions.
  • a mutant variant of Bag3 is a mutant Bag3 polypeptide as encoded by a BAG3 coding sequence including one or more of the SNPs associated to cardiomyopathy as described above, such as rs2234962 and rs3858340 or one of the SNP described in Table 1.
  • Specific antibodies, or antibody fragments, reactive against particular disease associated variant, for example, a particular Bag3 mutant polypeptide may be selected by screening expression libraries encoding immunoglobulin genes using phage display technologies.
  • antibodies are used to detect mutant Bag3 protein, in particular the mutant Bag3 protein as encoded by a B AG3 coding sequence including one or more of the SNPs associated to cardiomyopathy as described above, such as rs2234962 and rs3858340 or one of the SNP described in Table 1.
  • Said antibodies or fragments thereof bind to mutant Bag3 protein but not to wild type Bag3 protein.
  • said antibodies or fragments thereof bind to wild type Bag3 protein but not to mutant Bag3 protein.
  • Kits may be prepared for carrying out one of the above mentioned detection methods.
  • the invention further relates to a kit for carrying out the above-described method, said kit comprising: a. means for detecting a genetic mutation in BAG3 and/or HSPB7 gene locus, and, b. optionally, instructions for use of the kit.
  • kits for detecting a genetic mutation in BAG3 and/or HSPB7 gene locus may therefore comprises, specific primers or oligonucleotides probes as described above, or specific binding agents, such antibody or antibody fragments as described above.
  • the kits can include one or more isolated primers or primer pairs for amplifying a target nucleic acid in BAG3 or HSPB7 gene locus, such as a region comprising a SNP associated to cardiomyopathy as described above.
  • the kit can include primers for amplifying a haplotype including one, two, three, four, five S Ps in HSPB7 and/or BAG3, wherein the amplified sequence includes the SNP associated with cardiomyopathy.
  • the kit can further include one or more of a buffer solution, a conjugating solution for developing the signal of interest, or a detection reagent for detecting the signal of interest, each in separate packaging, such as a container.
  • the kit includes a plurality of size-associated marker target nucleic acid sequences for hybridization with a detection array.
  • the target nucleic acid sequences can include oligonucleotides such as DNA, RNA, and peptide-nucleic acid, or can include PCR fragments.
  • the kit includes binding reagent to disease associated polypeptide variant, such antibodies or fragment thereof.
  • the kit can also include instructions in a tangible form, such as written instructions or in a computer-readable format.
  • said kit comprises a. means for detecting one or more SNPs in BAG3 and/or HSPB7 gene locus selected from the group consisting rs 10927875, rs945417, rs2234962 and rs3858340; and, b. optionally, instructions for use of the kit.
  • said kit comprise a. means for detecting causal mutations in the full gene sequence of BAG3; and, b. optionally, instructions for use of the kit.
  • Figure 1 displays the association of these 14 S Ps with DCM in both the pooled and individual DNA analyses.
  • Figure 2 Ideogram from UCSC Genome Browser (assembly GRCh37/hgl9) presenting the associated region on chromosome 1 and gene structure and 5'->3' orientation. Positions of each of the five SNPs in LD defining DCM associated haplotypes are indicated.
  • DCM idiopathic dilated cardiomyopathy
  • conventional criteria 14 namely enlarged left ventricle diameter and low ejection fraction ( ⁇ 45%) associated with the absence of causal factors, such as coronary artery disease. Only apparently sporadic cases without affected first degree relatives were included.
  • GWAS genome wide association study
  • DCM cases were recruited through the CARDIGENE study (424 French patients) 42 , the EUROGENE (EHF) study (463 DCM cases from Germany, Italy and France) and the PHRC-DCM study (292 French DCM cases).
  • French controls were selected from the ECTFM Study (Etude Cas-Temoin sur rinfarctus du Myocarde) 43 and the FITENAT Study 44 . Controls were also selected from healthy consultants or hospital professional workers in clinical centres in Italy (72 controls) and Germany (278 controls). Selection of controls was stratified according to age, gender and geographic origin to match the distribution of DCM cases. Replications studies were conducted in two case-control studies, from Germany, 723 cases and 726 controls, and from United Kingdom, 442 cases and 576 controls. Constitution of DNA pools. The DNA pools were stratified on population as well as gender and age when the numbers permitted. We required that at least 25 samples were mixed in a single pool. Each pool was constituted twice and each pool replicate was analysed on two arrays independently.
  • the association between DCM and individual genotypes was tested using a logistic regression model (R:GLM) assuming an additive allele effect and adjusted on age, gender and study population.
  • R:GLM logistic regression model
  • DNAs from other available family members were genotyped for the familial variant by PCR and sequencing. We also sequenced exons 2, 3 and 4 of BAG3 in 364 individuals of European descent without known cardiac disease. Moreover, DNA from 95 controls of North African origin and 45 controls from Turkey were sequenced to check respectively for the presence of variants PI 15S and V468M in non-DCM populations.
  • the discovery GWAS was performed on pools of DNA (pools-GWAS). Overall 26 DNA pools were constituted according to study population, disease status, gender and age. To improve the precision of effect estimates when comparing pools of DNA from patients and controls, each pool was duplicated and each duplicate was hybridized to two different genotyping arrays. The allele quantification in pools was performed using the Illumina 610 quad beadchip ⁇ Illumina)). After filtering out copy number variation (CNV) markers and single nucleotide polymorphisms (SNPs) with poor signal intensity, we retained 517,382 SNPs in the analysis. Pools-GWAS, validation by individual DNA genotyping and replication
  • rsl0927875 is located in an intron of ZBTB17 (zinc finger and BTB domain containing 17 also frequently referred as MIZ-1) on chromosome Ip36.2-p36.1.
  • the gene is located within a region exhibiting strong linkage disequilibrium (LD) and spanning several other genes: SPEN (spen homolog, transcriptional regulator), HSPB7 (heat shock 27 kDa protein family, member 7), CLCNKA (chloride channel Ka) and CLCNKB (chloride channel Kb).
  • Yin/yang haplotypes To get better insight into the haplotypic structure of the region and its impact on the association of the locus with DCM, haplotypic relative risks 16 were computed.
  • the 5 genotyped SNPs determine 6 common haplotypes.
  • the two major haplotypes CTGCT (0.508 and 0.603 in controls and DCM cases, respectively) and TGCTA (0.301 and 0.230) differ at the five sites and are likely to represent ancestral alleles, the four less common haplotypes resulting from recombination between them.
  • HSPB7 is the best candidate at the locus but does not exhibit any coding variant.
  • HSPB7 that encodes the "cardiovascular heat shock protein” 23 and exhibit cardiac-specific expression
  • DCM cardiac-specific gene
  • HSPB7 expression is strongly affected by c/s-acting SNPs. Given the lack of coding variant in the HSPB7 sequence, we envisaged that sequence variations in the HSPB7 region might be related to DCM risk through an effect on gene expression. To investigate this hypothesis we looked for eQTLs in the HSPB7 region using two large eQTLs databases built from RNA expression data obtained from circulating monocytes (GHS Express 15 ) and from circulating monocytes and in v/Yro-derived macrophages (Cardiogenics Express 24 ). In both studies, HSPB7 mRNA was not detected in monocytes.
  • rs2234962 is a non-synonymous S P (c.T757C, p.C151R) located within the coding sequence of BAG3 on chromosome 10q25.2-q26.2.
  • S P c.T757C, p.C151R
  • a plot of DCM-association -values of SNPs located around rs2234962 shows that the other associated SNPs in the pools-GWAS exhibit rather modest -values in comparison to rs2234962 (data not shown); 3. as rs2234962 could tag functional SNPs absent from the genotyping array used in our study, we examined the LD of this SNP with other variants in the region and observed that only 2 SNPs in HAPMAP release 22 were in tight LD (r 2 > 0.80) with the lead SNP and both were intronic with no evidence that they could be functional.
  • BAG3 may affect DCM risk.
  • the HAPMAP database revealed that the BAG3 sequence presents several non-synonymous SNPs.
  • rs3858340 c.C1526T, p.P407L
  • rs35434411 c.G518A, p.R71Q
  • the 9 remaining variants were found once and none of them was present in the control group. All carriers of these mutations were heterozygous.
  • the identified variants included three insertions/deletions resulting in a truncation of the encoded protein sequence (Q251RfsX56, R396GfsX48, S385QfsX56), a substitution creating a premature stop codon (p.R309X) and 5 missense mutations leading to single amino acid changes (I94F, PI 15S, P380S, E455K, V468M).
  • Table 4 Identified SNPs and mutations in the coding sequences of BAG3 in 168 index cases with familial form of DCM Exo genomic variant name protein effect nature of dbSNP frequency frequency n position variant reference in index in controls, cases, n/694 n/336 alleles (%) alleles (%)
  • the six remaining variants affect the BAG3 coding sequence and are likely to be disease causative.
  • Two of the BAG3 mutations were observed in relatively large families (6 and 5 proven mutation carriers, respectively) and the 4 others were observed in small families (1 to 2 mutation carriers in each of these families).
  • the penetrance was high, with presence of DCM in 13/18 mutation carriers (72%) and possible DCM in 3 (LV dilatation and mild LVEF ⁇ 60% in 3, congestive heart failure in 2).
  • Two mutation carriers had a normal cardiac examination (a 7-year- old male and a 33-year-old female).
  • pools-GWAS also exhibit a reduced sensitivity, implying that our study may have missed some associations that would have been identified by an individual-based genotyping. Nevertheless, this approach is efficient ' ' and the interest of the Illumina technologies for pools-GWAS has been emphasized 26 .
  • the first DCM-associated SNP is located in a region exhibiting a yin/yang haplotype structure and encompassing the HSPB7 gene.
  • HSPB7 is also called cardiovascular HSP as a consequence of its selective expression in cardiovascular tissues 30 and it belongs to the small HSP (sHSP) family 31 , whose member's main function is to protect a variety of tissues by binding denatured proteins.
  • the physiological response of muscle fibers to stress involves HSPs of high molecular mass, such as HSP70 and sHSPs.
  • HSP5 ocB-crystallin
  • a dominant mutation in this gene causes a severe form of desmin-related cardiomyopathy characterized by accumulation of misfolded proteins as a consequence of impaired autophagy 32 .
  • BAG3 is a member of a conserved family of cyto-protective co-chaperones proteins containing a conserved domain able to interact with HSC70/HSP70 and sHSPs proteins. BAG3 is involved in numerous activities including macro-autophagic protein degradation in aging cells 34 . BAG3 is mainly expressed in striated muscle and colocalizes with Z-disks (a sarcomeric protein assembly essential for actin anchoring in striated muscles).
  • Bag-3 deficient mice Following normal muscle development, Bag-3 deficient mice present a progressive myopathy with Z-disk disruption and develop a fulminant myopathy characterized by noninflammatory myofibrillar degeneration with apoptotic features 35 . Autophagic degradation appears essential for maintaining Z-disk integrity and muscle contractility and Bag3 plays an essential role in this process which also involves small Hsps, such as HspB8 36 .
  • Bag3 knock-out mice fulminant myopathy and cardiomyopathy are observed only in homozygous -/- animals but not in heterozygous 35 while in drosophilia, the Bag3 ortholog starvin deletion is associated with locomotion decline and myofiber sarcomeric disorganization even in heterozygous animals 36 . Further experiments are clearly needed to better understand if either poison peptide or haplo-insufficiency drives the pathophysiology of DCM related to BAG3 mutations.
  • this GWAS identified two loci associated with sporadic DCM.
  • the most likely candidate genes at these loci are HSPB7 and BAG3.
  • BAG3 the two polymorphic loci in other forms of heart failure and muscle dysfunction, as well as in the age-related changes that affect the heart and muscle physiology.
  • drug targeting the "proteasis network" 36 ' 41 may interfere with the progressive increase in muscle weakness seen in DCM patients and elderly people.
  • An important aspect of this study is the discovery of BAG3 sequence variants being involved in both sporadic and familial forms of DCM. Table 5: Nucleotide sequences for practicing the method of the invention

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Abstract

L'invention concerne le domaine du diagnostic moléculaire, en particulier pour le procédé de diagnostic in vitro afin de détecter la prédisposition génétique d'un patient à la cardiomyopathie, telle que la cardiomyopathie dilatée. L'invention concerne plus particulièrement un procédé de diagnostic in vitro pour détecter la prédisposition génétique d'un patient humain à la cardiomyopathie, ce procédé comprenant les étapes consistant à : a. effectuer un prélèvement biologique sur un patient humain; et, b. détecter la présence ou l'absence d'une variante associée à la maladie HSPB7 et/ou d'une variante associée à la maladie BAG3 dans ledit prélèvement biologique, la présence de cette variante associée à la maladie HSPB7 ou BAG3 indiquant une prédisposition génétique du patient humain à la cardiomyopathie.
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IT201600069391A1 (it) * 2016-07-04 2016-10-04 Univ Degli Studi Di Salerno Uso della proteina bag3 e suoi frammenti peptidici per il controllo dell’omeostasi vascolare
EP3337518A4 (fr) * 2015-08-17 2019-06-12 Temple University Of The Commonwealth System Of Higher Education Compositions à base de bag3 et méthodes associées
CN110438159A (zh) * 2019-07-03 2019-11-12 广州中医药大学(广州中医药研究院) 一种引发肌原纤维肌病的基因突变小鼠模型的构建方法
CN110452979A (zh) * 2019-09-03 2019-11-15 郑州大学第一附属医院 一种影响人扩张型心肌病诊治的ttn g20137t突变及其应用
WO2019237002A1 (fr) * 2018-06-08 2019-12-12 Temple University - Of The Commonwealth System Of Higher Education Optimisation d'une thérapie génique de bag3
CN110863044A (zh) * 2019-12-11 2020-03-06 昆明理工大学 用于检测vcl基因突变的引物组合及其应用
EP3812473A1 (fr) * 2014-01-31 2021-04-28 Temple University Of The Commonwealth System Of Higher Education Bag3 comme cible pour le traitement de l'insuffisance cardiaque
CN116064780A (zh) * 2023-01-19 2023-05-05 中山大学附属第三医院 相关生物标记物在肝硬化心肌病中的应用

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