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WO1997011089A1 - Mutation genetique chez les patients atteints de cardiomyopathie idiopathique avec dilatation - Google Patents

Mutation genetique chez les patients atteints de cardiomyopathie idiopathique avec dilatation Download PDF

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WO1997011089A1
WO1997011089A1 PCT/US1996/014081 US9614081W WO9711089A1 WO 1997011089 A1 WO1997011089 A1 WO 1997011089A1 US 9614081 W US9614081 W US 9614081W WO 9711089 A1 WO9711089 A1 WO 9711089A1
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release channel
dilated cardiomyopathy
idiopathic dilated
seq
release
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PCT/US1996/014081
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English (en)
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Luyi Sen
Kenneth D. Philipson
Aldona Jake Lusis
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Regents Of The University Of California
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Priority to AU71537/96A priority Critical patent/AU7153796A/en
Publication of WO1997011089A1 publication Critical patent/WO1997011089A1/fr

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    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates generally to the field of genetic screening for inherited disease. More specifically, the invention regards a method of identifying individuals at risk of developing idiopathic dilated cardiomyopathy.
  • Idiopathic dilated cardiomyopathy formerly called congestive cardiomyopathy, is a syndrome characterized by cardiac enlargement and congestive heart failure. Although no etiology is definable in most cases, the congestive cardiomyopathy is believed to represent the result of myocardial damage caused by toxic, metabolic or infectious agents. Diagnosis of this disease depends solely on the exclusion of other possible causes at a late stage of the disorder. Nearly 40% of the patients receiving heart transplants suffer from idiopathic dilated cardiomyopathy.
  • SR sarcoplasmic reticulum
  • Ca ⁇ -ATPase expression is decreased in patients having end-stage heart failure caused by the various cardiomyopathies.
  • the present invention relates to treatment of idopathic dilated cardiomyopathy patients.
  • wild type SR CA ** release channel genes are introduced into patients bearing a mutant SR CA *+ release channel genes in a manner such that they are expressed to prevent development of idopathic dilated cardiomyopathy.
  • the present invention relates to the following aspects related to treatment and diagnosis of idopathic dilated cardiomyopathy patients:
  • the present invention is an isolated poiynucleotide encoding a mutant human SR Ca ** release channel.
  • This poiynucleotide comprises the sequence of SEQ ID NO:3 with the exception of having nucleotide positions 380 and 776 substituted by residues other than guanosine.
  • Nucleotide position 380 can be substituted by adenosine, and/or nucleotide position 776 substituted by thymidine.
  • Another aspect of the invention is a method of identifying an individual genetically predisposed to idiopathic dilated cardiomyopathy.
  • This method includes the steps of: obtaining a tissue sample from the individual, obtaining a population of polynucleotides from the tissue sample, determining if the population includes a poiynucleotide having the sequence of SEQ ID NO:3 except with a substitution at either position 380 or 776.
  • the individual is identified as being genetically predisposed to idiopathic dilated cardiomyopathy if the poiynucleotide is present.
  • the tissue sample can be any of a number of types of samples, such as a sample of blood or a sample of cardiac myocytes.
  • the determining step comprises PCR amplification and/or DNA sequencing. In certain cases, a substitution of adenosine at position 380 of SEQ ID NO:3 or a substitution of thymidine at position 776 of SEQ ID NO:3 is identified.
  • the determining step comprises cleavage by a restriction endonuclease, such as Hindlll followed by electrophoresis. In this embodiment, cleavage can sometimes result in the formation of two poiynucleotide fragments, such as fragments having lengths of from between about 300 and 800 base pairs.
  • the determining step comprises identifying a 3.7 kb Hindlll restriction fragment length polymorphism, wherein the presence of the fragment indicates the presence of the poiynucleotide.
  • a further aspect of the present invention is a pair of oligonucleotides primers for amplifying a segment of the SR Ca ** release channel gene.
  • a first ofthe pair is homologous to a sequence contained within SEQ ID NO:4 on one side of a region including a Hindlll restriction endonuclease cleavage site at position 777 of the sequence of SEQ ID NO:4, and a second of the pair is homologous to a sequence contained within SEQ ID NO:4 on another side of the region.
  • the pair of primers can have the sequences SEQ ID NO:1 and SEQ ID NO:2 or SEQ ID NO:5 and SEQ ID NO:2.
  • Yet another aspect of the invention is an immunohistochemical method of identifying an individual at risk of idiopathic dilated cardiomyopathy.
  • This method includes the step of obtaining first and second antibody reagents.
  • the first antibody has binding specificity for a wild-type SR Ca ** release channel
  • the second antibody has binding specificity for a mutant SR Ca ** release channel.
  • the mutant SR Ca ** release channel is expressed in individuals afflicted with idiopathic dilated cardiomyopathy.
  • the method also includes the steps of obtaining from the individual a tissue sample containing cardiac myocytes, testing the myocytes for staining by the first and second antibody reagents, and identifying individuals at risk of idiopathic dilated cardiomyopathy as those individuals whose myocytes were stained by the second antibody but not by the first antibody.
  • Still another aspect of the present invention is a lipid bilayer having inco ⁇ orated therein a substantially purified mutant SR Ca ** release channel protein obtained from a mammal genetically predisposed to idiopathic dilated cardiomyopathy.
  • the substantially purified mutant SR Ca *+ release channel protein can be derived from sarcoplasmic reticulum membranes isolated from myocardium membranes.
  • One more aspect of the invention is a method of identifying drugs useful in the treatment of individuals afflicted with idiopathic dilated cardiomyopathy.
  • This method includes the steps of (a) preparing a first lipid bilayer having incorporated therein SR Ca ** release channels substantially purified from cardiac myocytes obtained from an individual afflicted with idiopathic dilated cardiomyopathy and (b) preparing a second lipid bilayer having incorporated therein SR Ca ** release channels substantially purified from cardiac myocytes obtained from an individual not afflicted with idiopathic dilated cardiomyopathy.
  • An assay is performed to measure SR Ca ** release channel gating properties of the first and second bilayers in the absence and presence of a test drug. The results of the assay in the absence and presence of the test drug are compared. Drugs are identified as useful in treating individuals afflicted with idiopathic dilated cardiomyopathy as a test drug which causes causing mutant
  • An additional aspect of the present invention relates to a transgenic mouse having a genome that includes at least one copy of a poiynucleotide encoding a mutant SR Ca ** release channel.
  • the poiynucleotide encoding a mutant SR Ca ** release channel can have point mutations characteristic of humans afflicted with idiopathic dilated cardiomyopathy, such as point mutations included in the sequence of SEQ ID NO:3 where nucleotide positions
  • nucleotide position 380 and 776 are substituted by residues other than guanosine.
  • nucleotide position 380 is substituted by adenosine and nucleotide position 776 is substituted by thymidine.
  • Still another aspect of the present invention relates to a transgenic hamster having a genome comprising a Bio 14.6 strain hamster genome and a transgene encoding a wild-type SR Ca ** release channel.
  • the wild-type SR Ca ** release channel can be a human wild-type SR Ca ** release channel, or a wild-type SR Ca *+ release channel derived from a hamster other than a Bio 14.6 strain hamster.
  • Yet one more aspect of the present invention is a method of alleviating symptoms associated with idiopathic dilated cardiomyopathy in a patient.
  • This method involves providing heart cells of the patient with a poiynucleotide encoding a functional SR Ca ** release channel.
  • a viral expression vector physically linked to the wild-type poiynucleotide encoding the SR Ca ** release channel can be used for this purpose.
  • Suitable viral expression vectors include a retroviral expression vector, an SV40-based expression vector, an adenoviral expression vector, a herpes viral expression vector, an adeno-associated viral expression vector, a vaccinia viral expression vector and a bovine papillomavirus expression vector.
  • the functional SR Ca ** release channel can have the sequence of SEQ ID NO:3.
  • a catheter is used for delivering the viral expression vector to the heart cells.
  • Figure 1 shows the contractile amplitude of isolated cardiac myocytes from the cardiomyopathic hamster hearts at prehypertrophic, hypertrophic and heart failure stages and age-matched normal hamster hearts.
  • A Superimposed are the amplitude of cell motion traces recorded from a hypertrophic myocyte (CM) and an age-matched normal myocyte (NH). The resting cell length for CM was 178 ⁇ m and NH was 154 ⁇ m.
  • B Means and
  • Figure 2 shows 45 Ca ** uptake by cardiac myocytes isolated from cardiomyopathic hamster hearts at three developmental stages and age- matched normal hearts. Values shown are mean ⁇ S.D. for cardiomyopathic cells (filled symbols) at prehypertrophic (triangles), hypertrophic (circles) and hearts failure (squares) stages and age-matched normal cells (filled symbols). Each point is the mean of data from 10 experiments performed in triplicate. * p ⁇ 0.01.
  • FIG. 3 shows [Ca ** ], transient in cardiac myocytes from cardiomyopathic hamster hearts at three developmental stages and age- matched hamster hearts.
  • [Ca ** ] transient associated with single contraction was examined using fura-2 fluorescence. Cells were electrically driven at 1.5 Hz using platinum electrodes. Superimposed are the [Ca ** ], transients recorded from a cardiomyopathic cell (CM) at heart failure stage and an age-matched normal cell (NH). Values shown are mean ⁇ S.D. for 18 cells from cardiomyopathic hamster hearts (hatched bars) at each age-groups and 18 cells from age-matched normal hamster hearts (open bars). * p ⁇ 0.01.
  • Figure 4 shows the contractile response to caffeine.
  • Isolated ventricular myocytes from 130-170 days old normal (open circles) and cardiomyopathic (filled circles) hamsters were electrically stimulated at 1.5 Hz while superfused with HEPES-buffered medium (pH 7.35, 37° C) containing 0.9 mM Ca ** . After equilibration, cells were perfused with Na+ - and ⁇ Ca ** - free HEPES buffer to assess SR Ca ** stores. Data are plotted as a percentage change of the contractile amplitude from control condition. Each point represents the mean ⁇ S.D. of 16 experiments using cells from 6 normal and 6 myopathic hearts.
  • Figure 5 shows the results of Ca ** release studies.
  • A The ATP-dependent SR Ca ** uptake as determined by extravesicular fura-2 signals.
  • Samples were from normal hamsters (o), and cardiomyopathic hamsters ( ⁇ ).
  • B The time course of caffeine-induced change in [Ca ** ] SR at two developmental stages. Samples were from normal hamsters at 30-40 days (o) and 200-300 days ( ⁇ ), and cardiomyopathic hamsters at 30-40 days (•) and 200-300 days (A).
  • Figure 6 shows the channel mean open time and channel mean closed time for normal and myopathic cells at the prehypertrophic stage.
  • A Long closed times. Open bars represent normal myocytes; closed bars represent prehypertrophic myopathic myocytes.
  • B Short closed times. Open bars represent normal myocytes; closed bars represent prehypertrophic myopathic myocytes.
  • idiopathic dilated cardiomyopathy at an early stage following disease onset allows for more specific treatment of the disease. Rather than providing a general treatment of the disease symptoms until the time of heart failure, progress of the disease could be slowed. The patient's iifespan will certainly be extended as the result of an early diagnosis and intervention. However, until now no test was available for making such an early diagnosis.
  • the nucleotide sequence of the human homolog has not been published and is not on deposit in a publicly accessible data base.
  • poiynucleotide sequences representing the human and rabbit SR Ca ** release channel genes are highly similar.
  • the point mutations at positions 380 and 776 of the cloned sequence presented as SEQ ID NO:4 correspond to positions 8366 and 8762 of the cDNA sequence disclosed by Otsu et al.
  • Example 1 describes the method used to isolate the ventricular myocytes that were used to study calcium handling. Myocytes were prepared from cardiomyopathic hamsters at prehypertrophic, hypertrophic, and heart failure stages.
  • cardiomyopathic and FIB healthy control hamsters Male Bio 14.6 cardiomyopathic and FIB healthy control hamsters were obtained from Biobreeders (Fitchburg, MA). The animals were obtained at developmental stages representing the prehypertrophic (20 - 30 days old), hypertrophic (130 - 170 days old) and heart failure stages (440 -480 days old). Both cardiomyopathic and the healthy control hamsters were maintained under the same conditions on a normal laboratory animal diet and tap water ad libitum.
  • ventricle was removed from the perfusion set-up, cut into 2 mm 3 pieces and placed in a flask containing 0.05% collagenase, 0.03% hyaluronidase, 0.001% trypsin and 1.0 mM CaCI 2 in Krebs-
  • tissue fragments were shaken (100 cycles/minute) in an orbital shaking water bath at 37°C. After 15 minutes, the tissue pieces were transferred into a Ca ** -free buffer containing 0.05% collagenase and 0.03% hyaluronidase. The tissue pieces were triturated 5 minutes using a 5 ml plastic pipette with a 6 mm orifice. Warm (37°C) oxygenated Ca ** - free buffer was added to the tube, and the cells were centrifuged at 400 ⁇ m (41 x g) for 1 minute.
  • the pelleted cells were then washed twice, using the same method, to remove the collagenase and hyaluronidase enzymes, isolated myocytes were resuspended in 0.6 mM Ca ** solution. Approximately 80% of cells exhibited rod-shaped mo ⁇ hology with clear cross-striations. Cells isolated according to the method described above were used to assess the contractile properties of myocytes obtained from hearts representing the various stages of disease progression. More specifically, the amplitudes of cell motion were measured in ventricular myocytes isolated from cardiomyopathic hamsters at the prehypertrophic stage (20 - 30 days old), hypertrophic (130 - 170 days old) or heart failure stage (440 - 480 days old) of development. As detailed below, comparisons were made with age and sex- matched normal controls (FIB).
  • FIB age and sex- matched normal controls
  • Example 2 describes the methods used to quantitate the contractile properties of ventricular myocytes.
  • the TV camera had an interlace defeat producing an image composed of 262 raster lines.
  • the motion detector monitored a selected raster-line segment and provided the amplitude of cell moving along the raster line at a sampling interval of 16 ms.
  • Light-dark contrast at the edge of the cell provided a marker for measurement of the amplitude of motion.
  • the analog tracing was recorded with a strip charge recorder. Cells chosen from experiments always contracted from an attachment point at the center of the cell. Both freely moving ends of the cell shortened with stimulation and the amplitude of the cell motion was same at both ends.
  • Example 3 describes the methods used to assess calcium flux in myocytes derived from cardiomyopathic and control hamster hearts.
  • Example 3 Analysis of Calcium Flux in Mvocvtes The procedure for measuring the 4S Ca ** uptake rate was essentially that described by Barry et al., in J. Physiol. 325:243 (1982). Isolated cells were prepared simultaneously from cardiomyopathic and control hamster hearts, suspended in 2 ml of Krebs-Hanseleit buffer with 20% albumin and allowed to settle for 5 minutes through the albumin at 37°C. The supernatant was then removed, and the sedimented viable cells were resuspended in 37°C HEPES- buffered media containing 0.9 mM Ca ** . Cells from either preparation were about 95% rod shaped.
  • Protein content was measured by standard methods using bovine serum albumin as a standard.
  • transient [Ca ** ] was measured in three stages of myopathic myocytes and age-matched normal control cells.
  • Example 4 describes the methods used to measure intracellular Ca ** concentrations.
  • Example 4 Measurement of Intracellular Ca ** Concentration [Ca ** ], of single cardiac myocytes obtained from cardiomyopathic and control hamsters were measured using the Ca ** -sensitive fluorescent dye, fura- 2.
  • Cells attached to glass coverslips were incubated with 3 ⁇ W ⁇ fura-2. AM for 10 minutes at room temperature and then washed for 5 minutes in HEPES- buffered medium to remove extracellular and bound dye.
  • the glass coverslip with attached cells was placed in a perfusion chamber specifically designed to fit the stage of a phase-contrast microscope (Nikon Inc., Garden City, NY) and perfused with oxygenated HEPES-buffered medium warmed to 37°C.
  • the microscope with a 40x objective was attached to a SPEX-fluorolog 2 instrument with excitation wavelengths set at 340 nm and 380 nm and emission wavelength set at 505 nm.
  • the two excitation wavelengths were made to alternate once every second for time-averaged [Ca ** ], measurements or 100 times per second for transient [Ca* * ], measurements and were stored in separate memories of an SPEX Datamate microcomputer (SPEX Industries, Inc., Edison, NJ). Cells chosen for [Ca ** ], measurement were electrically stimulated at 1.5
  • Chem. 260:3440 (1985) was used to transform the 340/380 nm fluorescence intensity ratios into [Ca ** ], values. Notably, use of the ratio (340/380 nm) compensated for variations involving dye concentration, dye leakage and cell thickness. Under the conditions of our studies, 3 ⁇ M fura-2 exposure for 10 minutes reduced the amplitude of cell motion by approximately 115%. Washing the cells produced no further decline in cell motion. Any cell in which fura-2 decreased the amplitude of cell motion by more than 30% was not used.
  • Example 5 describes the methods used to examine the effect of caffeine on SR channel function in 130-170 day old normal and cardiomyopathic hamster myocytes.
  • Example 5 Inotropic Response to Caffeine Cardiac myocytes isolated from hypertrophic and age-matched normal hamsters, prepared as in Example 1, were perfused with Na * -free HEPES buffer for 5 minutes, and then exposed to 20 mM caffeine in Na * - and Ca ** -free HEPES buffer to assess SR Ca * * content without the presence of Na * -Ca ** exchange.
  • the time- course of the effect of the Na * -free buffer alone (0 - 4 minutes) was similar in myopathic and normal cells.
  • SR Ca ** release channel distinguished the normal and disease-prone animals.
  • Example 6 describes the methods used to compare the SR Ca ** release channel functions in Bio 14.6 hamsters at the prehypertrophic stage, at the heart failure stage and in age-matched normal controls.
  • SR vesicles were prepared from cardiomyopathic and normal hamster hear ⁇ s nt sg ⁇ . . 30 ( 40 ** ja ⁇ s and 200 J .o 300 -jays ac-i -i ⁇ ng to the method described by Meissner et al., in J. Biol. Chem. 262:3065 (1987). Homogenates were initially subjected to differential centrifugation. The subtraction of cardiac SR vesicles was then isolated using a sucrose step density gradient. The free Ca ** activity inside SR vesicles was determined by measurement of the fluorescence ratio (340/380 nm) in fura-2-loaded SR vesicles.
  • Fura-2 was also used to monitor ATP-dependent Ca ** uptake by SR vesicles isolated from the cardiomyopathic and normal hamster hearts. Fura-2 acid (3 ⁇ M) was added outside the vesicles (5 ⁇ g) to buffer (2 ml) containing 500 ⁇ M ATP. Changes in fura-2 fluorescence resulted from the decline in free Ca ** during active Ca * * accumulation by the vesicles (Kargacin et al., Am J.
  • Figure 5a presents the Ca ** uptake by SR from myopathic hearts (CM) and normal hearts (NH) at the prehypertrophic stage.
  • CM myopathic hearts
  • NH normal hearts
  • the velocities of ATP-dependent Ca ** uptake by isolated SR vesicles was similar at both developmental stages in SR vesicle preparations from myopathic and normal hearts.
  • Figure 5b shows the velocities of Ca ** release estimated from the changes in the fura-2 fluorescence ratio produced by caffeine and ryanodine.
  • the velocity of Ca ** release by caffeine as judged by the intravesicular free Ca ** signal was significantly reduced in myopathic SR vesicles compared to normal controls at both early and late heart failure stages. Similar results have been obtained using ryanodine.
  • This abnormality in SR Ca ** release detectable at an early stage of this disease, may play an important role in contractile dysfunction.
  • the decrease of SR Ca ** release that accompanied normal SR Ca ** uptake may contribute to SR Ca ** overload at subsequent stages.
  • Example 7 describes the methods used to prove that the SR Ca* * Release Channel was altered in prehypertrophic myocytes.
  • Ca * * concentration range has also been compared in the SR vesicles from Bio 14.6 hamster hearts at the prehypertrophic stage and from age-matched normal hamster hearts using the method described above.
  • concentration-response curve of relative channel open probability in SR from myopathic heart was significantly shifted to the right.
  • concentration-response curve was significantly shifted to the left.
  • the area corresponding to the channel-active "window" was 67% decreased in prehypertrophic cells when compared with age-matched normal cells. This alteration was also found in hearts isolated from 15 to 18 day old prehypertrophic hamsters.
  • Example 8 describes the methods used to identify a difference between the structures of the SR Ca * * release channel genes of hamsters that were normal controls or that were genetically predisposed to the development of cardiomyopathy.
  • Genomic DNA was prepared from normal (FIB) and cardiomyopathic (Bio 14.6 and Bio 53.58) hamster hearts. Tissue was rapidly frozen and crushed to produce readily digestible pieces. The processed tissue was placed in a solution of proteinase K and sodium dodecyl sulfate and incubated until most of the cellular protein was degraded. The digest was deproteinized by successive phenol/cloroform/isoamyl alcohol extractions, recovered by ethanol precipitation, dried and resuspended in TE buffer (Enrietto et al., Cell 35:369 (1983)).
  • Southern analysis was carried out according to standard methods. Briefly, 10 ⁇ g samples of genomic DNA were digested with 18 different restriction endonucleases and then separated by agarose gel electrophoresis.
  • the restriction enzymes employed in this procedure were: EcoRI, BamHI, Neil, Apa1, Kpnl, Pstl, BssHII, Mbol, Bell, Notl, Ddel, Spel, Aval, Taql, Seal, Sacll,
  • the gels were then blotted to a nylon membrane.
  • the SR Ca ** release channel probe was random primed using a commercially available kit (Pharmacia).
  • the cDNA encoding the human cardiac muscle ryanodine receptor used for this preliminary study was provided by Dr. A. Marks' laboratory. This cDNA probe, called HCRC1, corresponded to nucleotides
  • the membrane was washed under standard high stringency conditions and exposed to X-ray film at -70°C with an intensifying screen.
  • genomic DNA isolated from peripheral blood lymphocytes could serve as a source of genomic DNA that can be tested for the presence of the genetic indicator of susceptibility to idiopathic dilated cardiomyopathy.
  • Example 9 describes the methods used to identify an RFLP that was associated with idiopathic dilated cardiomyopathy in humans.
  • HCRC1 Human Cardiac Release Channel 1
  • This cDNA probe corresponded to nucleotides 4460-5440 of the rabbit cardiac Ca ** release channel cDNA (Brillantes et al., Circulation Research 71:18 (1992)), and was synthesized alternatively by nick translation or random priming with equally good results. Following hybridization and washing under high stringency conditions, all according to standard protocols, the membranes were air dried and autoradiographed.
  • RFLP represented by a 3.7 kb DNA fragment
  • the HCRC1 cDNA probe identified a 3.7 kb band in Southern blotted genomic DNA from individuals afflicted with idiopathic dilated cardiomyopathy.
  • This 3.7 kb band was absent from the digests of genomic DNA isolated from control individuals. This finding led us to investigate the nature of the mutation that gave rise to the
  • Example 10 describes the method used to identify the mutation within the SR Ca * * release channel gene that produced the 3.7 kb RFLP associated with idiopathic dilated cardiomyopathy.
  • Hindlll digested genomic DNA isolated from patients afflicted with- idiopathic dilated cardiomyopathy was size fractionated on 0.8 % agarose.
  • a GENE-CLEAN KIT (BIO 101) was used to purify DNA from the region ofthe gel having DNA fragments 3.7 kb in length. These fragments were subsequently cloned into the pUCNC vector using the PCR-CLONING SYSTEM (5'-3' Inc.). A colony hybridization protocol using radiolabeled HCRC1 probe was used to identify clones harboring the desired fragments.
  • Dideoxy nucleotide sequencing of the cloned 3.7 kb fragment was performed using SEQUENASE 2.0 (U.S. Biochemical) and [or ⁇ SJdATP (Amersham and DuPont-NEN). To produce all of the necessary sequencing templates, unidirectional deletions were prepared using the ERASE-A-BASE system from Promega.
  • the cloned fragment was found to correspond to nucleotides 4650-8760 of the cDNA encoding the human cardiac Ca ** release channel reported by Tunwell et al., in Biophysical Journal 68:A52 (1995).
  • a point mutation was discovered in 10/10 patients with idiopathic dilated cardiomyopathy.
  • Codon 8258 was mutated from AGG to AAG, resulting in an amino acid change from arginine to lysine.
  • there was a missense mutation at codon 8634 that resulted in an amino acid change from glutamine to histidine.
  • a simple PCR protocol was employed to amplify a genomic DNA fragment that encompassed the region of the SR Ca ** release channel gene that distinguished the normal and the idiopathic dilated cardiomyopathy genotypes. More specifically, oligonucleotide primers were selected for use in a procedure that would amplify the genomic
  • Example 11 describes the methods used to determine whether genomic DNA encoding the SR Ca ** release channel gene of patients with ischemic cardiomyopathy exhibited the same mutation we had identified in patients afflicted with idiopathic dilated cardiomyopathy.
  • Genomic DNA was isolated from ventricular muscle tissue of patients diagnosed with ischemic cardiomyopathy according to standard laboratory procedures. Oligonucleotide primers having the sequences 5'-
  • TTCAAACTGGCACTGCCTTGCCTGAGTGCCGTTGC-3' (SEQ ID NO:1) and 5'-AAGTTTGCAGAATAGGCTAGTCACCATTTC-3' (SEQ ID NO:2) were used as primers in a standard PCR protocol. These primers -corresponded to nucleotides 7987-8021 and 9007-9036 of the rabbit SR Ca ** release channel gene, respectively. Conditions used in the PCR amplification included 35 cycles of: 90°C for 30 seconds, 42°C for 30 seconds and 70°C for 60 seconds. The buffer used in the amplification procedure has been described by Sidransky et al., in Science 252:706 (1991).
  • the -1.1 kb amplification products were cloned into T-tailed plasmid vectors using the PCR CLONING SYSTEM from 5'-3', Inc.
  • the cloned inserts were then sequenced using a modified T7
  • the nucleotide sequence of the cloned DNA fragment derived from patients having ischemic cardiomyopathy is presented as SEQ ID NO: 1
  • the first approach involved DNA sequence analysis to determine whether a patient's DNA sample contained either an adenosine residue at position 380, or a thymidine residue at position 776 of the SR Ca ** release channel gene segment represented by the wild-type DNA sequence of SEQ ID NO:3. As will be apparent from the preceding disclosure, these two nucleotide positions represent the differences that distinguish the SR Ca ** release channel gene segments represented by SEQ ID NO:3 and SEQ ID NO:4. When either mutation was present in the patient's DNA sample, the patient was identified as at-risk for idiopathic dilated cardiomyopathy.
  • cleavage of an amplification product that encompassed the position corresponding to the Hindlll site within a DNA fragment represented by the sequence of SEQ ID NO:4 indicated the presence of the mutation that correlated with susceptibility to idiopathic dilated cardiomyopathy.
  • DNA sequencing of that amplification product could be carried out to verify the absence of the mutation that conferred susceptibility to idiopathic dilated cardiomyopathy. Such a procedure would provide one means of ensuring against false negative results.
  • Example 12 describes an assay for detecting a Hindlll cleavage site that was diagnostic of idiopathic dilated cardiomyopathy.
  • Example 12 Genetic Test for Idiopathic Dilated Cardiomyopathy - Genomic DNA was extracted from tissues of 10 idiopathic dilated cardiomyopathy patients, 4 ischemic cardiomyopathy patients and 4 normal controls according to standard procedures.
  • the 10 idiopathic dilated cardiomyopathy patients used in this Example represented 10 of the 18 individuals who possessed the 3.7 kb RFLP in the procedure of Example 9.
  • Cardiac myocytes served as the tissue source for genomic DNA isolated from the cardiomyopathy patients while peripheral blood lymphocytes served as the source of DNA for the normal control samples.
  • a fragment of genomic DNA corresponding to the span from 7987 to 9036 of the rabbit SR Ca ** release channel gene was amplified by a PCR protocol that employed two oligonucleotide primers having the sequences of SEQ ID NO:1 and SEQ ID NO:
  • Genomic DNA samples (100 ng) were used as templates in 25 ⁇ l reactions that contained 10 mM Tris-HCI (pH 8.3), 50 mM KCI, 0.75 mM MgCI 2 , 0.01% gelatin, 200 ⁇ M dNTPs, 1 ⁇ M each oligonucleotide primer and 5 units of Taq DNA polymerase.
  • PCR conditions consisted of 35 cycles of 95°C for 30 seconds, 42°C for 30 seconds, and 70°C for 60 seconds.
  • nucleotide positions 230 and 626 of this amplification product represented the two nucleotides that were diagnostic of idiopathic dilated cardiomyopathy.
  • Hindlll cleavage of the poiynucleotide represented by the sequence' of SEQ ID NO:6 resulted in cleavage products of approximatly 0.6 kb and 0.3 kb.
  • primer sets can be used to amplify segments of the SR Ca ** release channel gene that encompass either or both of the nucleotide positions disclosed to identify individuals at risk of idiopathic dilated cardiomyopathy.
  • the products of those amplification reactions can then be used in DNA sequencing -protocols or Hindlll cleavage protocols, as described above, to create a diagnostic test for idiopathic dilated cardiomyopathy.
  • Example 13 describes an immunohistochemical test that can be used to identify individuals at risk of developing idiopathic dilated cardiomyopathy.
  • An Immunohistochemical Test for Idiopathic Dilated Cardiomyopathy An antigenic composition comprising a segment of the SR Ca ** release channel representing the domain of the protein that is mutant in idiopathic dilated cardiomyopathy is first prepared.
  • the composition is likely a fusion protein, but may also be a synthetic peptide and may be coupled to a carrier for improved antigenicity.
  • a monoclonal antibody, or variant thereof, having binding specificity for the mutant form of the SR Ca ** release channel is next prepared according to methods that will be appreciated by those having ordinary skill in the art.
  • the mutant-binding antibody can specifically bind the mutant form of the receptor without binding the wild-type receptor.
  • a tissue sample containing cardiac myocytes is obtained by standard methods from an individual to be tested for genetic susceptibility to idiopathic dilated cardiomyopathy.
  • the method of obtaining the myocytes may involve the use of a catheter.
  • the sample is next prepared for analysis by immunohistology using the two antibody preparations as reagents. If the wild- type-binding antibody stains the tissue sample, the tissue donor is either not susceptible, or is an unaffected carrier of the trait conferring susceptibility to idiopathic dilated cardiomyopathy. If the mutant-binding antibody stains the tissue sample, and the wild-type-binding antibody does not stain, the tissue donor is susceptible to idiopathic dilated cardiomyopathy.
  • This immunohistological assay represents yet another method that can be used in a diagnostic procedure for identifying individuals susceptible to idiopathic dilated cardiomyopathy.
  • the mutant form of the SR Ca ** release channel disclosed herein is useful in assays to identify candidate therapeutic agents for the treatment of idiopathic dilated cardiomyopathy. More specifically, we anticipate that drugs which cause the mutant SR Ca ** release channel to behave like the wild-type channel are candidates for the treatment of idiopathic dilated cardiomyopathy. We believe that these anticipated drugs will increase the amount of calcium released from the SR in the excitation-contraction coupling process. This would effectively increase the myocardium contraction.
  • Example 14 describes in vitro methods that can be used to identify drugs that cause mutant SR Ca * * release channels to behave like wild-type channels. Drugs identified by the following method can then be tested in vivo as therapeutics by methods well known to those of ordinary skill in the art for effectiveness in the treatment of idiopathic dilated cardiomyopathy.
  • Example 14 Drug Discovery Using Mutant SR Ca* * Channel Protein Myocardial SR is fractionated into heavy, intermediate, and light density vesical fractions by differential and sucrose gradient centrifugation as described previously. Heavy SR membranes containing the Ca ** release channel are recovered from the 36-45% region of a sucrose gradient that contained myocardium membranes.
  • Muller-Rudin planar bilayer containing phosphatidytanoylphos- phatidylcholine (10 mg/ml) in decane is painted across a 200 ⁇ m hole in a
  • the cis chamber is defined as the side to which SR vesicles are added; the opposite side is referred to as the trans chamber. All additions of Ca ** , Mg ** , EGTA or adenine nucleotides are made to the cis chamber. Applied voltages are defined with respect to the trans chamber held at virtual ground and therefore agree with the normal cellular convention.
  • the cis chamber, inside the Lexan cup contains 0.25 M choline Cl, 5 mM CaCI 2 , 100 ⁇ M EGTA, 10 mM Tris HEPES, pH 7.4 and the trans chamber contains 50 mM choline Ca, 5 mM CaCI 2 , 100 ⁇ EGTA, 10 mM Tris HEPES, pH 7.4.
  • Heavy SR vesicles in 0.3 M sucrose, 10 mM K Pipes (1,4-piperazinediethanesulfonic acid), pH 7.0, are added to the cis chamber and stirred (final protein concentration, 3 ⁇ g/ml). Shortly after vesicle addition, step-like vesicle-bilayer fusion events are observed.
  • both chambers are perfused to remove any permeate anions and unfused vesicles.
  • the trans chamber is perfused with 3 vol of 52 mM
  • Tris/HEPES (pH 7.4).
  • buffer is pumped into the bottom of each chamber via a small Tygon hose and simultaneously withdrawn through a hose positioned at the top of the chamber.
  • the density of the HEPES perfusion buffer is greater than that of the choline Cl solution so that during perfusion the choline Cl is effectively displaced by the HEPES solution.
  • Single channel currents are recorded and voltage control is imposed with a commercial patch clamp unit (Axon Instruments, Foster City, CA).
  • the data is acquired in real time and filtered at a cut-off frequency of 100 Hz with an 8- pole Beisel filter, digitized at 500 Hz and stored for analysis.
  • the ionic currents are subjected to conventional single channel analysis, carried out using the pCLAMP software system (Axon Instruments, Foster City, CA) and custom programs.
  • the analysis can be started with amplitude histograms to determine the single channel conductance. Once the average single chan ⁇ el current is determined, opening and closing transitions can be detected using as threshold criterion a level equal to 0.5 of the predominant open channel current.
  • Channel gating properties include the single channel open probability, open and close lifetime, single channel current voltage relation and channel kinetics. These properties can be compared between vesicles from normal organ donors and the heavy SR vesicles from myocardium of patients with end-stage heart failure caused by various cardiomyopathies
  • Drugs to be tested for their effects on SR Ca ** release channel properties can be added to the cis chamber for bilayers that inco ⁇ orate vesicles obtained from cardiomyopathic myocytes. Readings can be obtained before and after drug addition. Drugs that cause SR Ca * * release channels from cardiomyopathic myocytes to exhibit properties similar to SR Ca ** release channels isolated from normal control myocytes are candidates as therapeutic agents in the treatment of idiopathic dilated cardiomyopathy.
  • nucleic acid-based diagnostic testing additionally indicated that idiopathic dilated cardiomyopathy patients were homozygous for the mutant SR Ca ** release channel gene. More specifically, we discovered that PCR amplification using genomic DNA templates isolated from idiopathic dilated cardiomyopathy patients gave rise to products that harbored SR Ca ** release channel-encoding polynucleotides having a novel Hindlll cleavage site at position 776. Since all amplification products in reactions performed using affected patient DNA templates harbored a point mutation diagnostic of the disease, we concluded that no wild-type templates were present in the starting sample of patient DNA.
  • At least two approaches that employ animal models can be used to confirm that homozygosity for a mutant SR Ca ** release channel gene is causative in the development of idiopathic dilated cardiomyopathy.
  • a line of transgenic Bio 14.6 hamsters that harbor a recombinant expression vector that directs expression of a wild-type SR Ca ** release channel protein can be created.
  • control Bio 14.6 hamsters develop idiopathic dilated cardiomyopathy by approximately 400 days of age
  • transgenic animals expressing a wild-type SR Ca ** release channel are not expected develop such symptoms if disease development is inhibited by the presence of at least one wild-type copy of the SR Ca ** release channel gene in the genome.
  • Viral vectors useful in the practice of the present invention include those that are based on viruses capable of infecting mammalian cells. These viruses use receptor- mediated pathways for the delivery of foreign genetic elements to target cells.
  • viral vectors have specific mechanisms for the expression of their genomes, either through maintenance of episomal elements or by integration into the host cell genome.
  • Viral vectors are generally constructed with deletions in the virus genome that render them replication incompetent in the absence of helper or wild-type viruses.
  • the complementing functions ofthe helper viruses can in some cases be fully substituted by replication-defective expression units that provide trans-acting viral proteins necessary for vector assembly and transduction.
  • retroviral vectors are highly efficient at transducing only dividing cells, it is prefered that nonretroviral vectors be used for direct transfer of a therapeutic gene into adult cardiac myocytes in vivo.
  • retroviral vectors may be employed.
  • Preferred viral vector systems presently used for introducing foreign genes into mammalian cells include: SV40, adenovirus, he ⁇ es virus, adeno-associated virus, vaccinia, and bovine papillomavirus. Both adenovirus (Karlsson et al. EMBO J. 2:2377 (1986)) and adeno-associated virus (La Face et al., Virology 162:483 (1988)) vectors have been used to transduce marrow-derived cell lines.
  • a number of different techniques can be employed for delivering a therapeutic gene composition to the heart of an individual receiving gene therapy.
  • one technique involves direct injection of a therapeutic SR Ca ** release channel expression vector into the cardiac muscle through right or left ventricular catheterization.
  • Another technique involves administration of a therapeutic SR Ca ** release channel expression vector into the coronary artery via coronary artery catheterization.
  • a cardiac catheter can be inserted into the heart, and a composition comprising a vector operably linked to an SR Ca* * release channel-encoding poiynucleotide can be introduced into the heart through the catheter.
  • a catheter delivery system it may be advantageous to prevent the heart from contracting during the time the therapeutic composition is desired to be in contact with the heart tissue. In this way, the opportunity for introducing the therapeutic construct into the cells of the heart may be enhanced.
  • Cis-regulatory transcriptional regulatory and RNA processing signals of the therapeutic receptor-encoding poiynucleotide will be under the control of vector-borne gene sequences. These sequences include a promoter for initiating transcription of the poiynucleotide and a polyadenylation signal sequence. An intron may also be present in the vector sequences and may appear in the primary transcript. Cis-regulatory elements which include enhancer elements may also be present in the expression vector and may serve to modulate promoter activity. In aggregate, the promoter and enhancer are expected to exhibit substantially constitutive expression of the linked receptor-encoding poiynucleotide. However, we also contemplate that inducible promoters and/or enhancers will be useful in controlling transcriptional expression of the linked therapeutic gene sequence.
  • transgenic technology can be employed to prove that idiopathic dilated cardiomyopathy is attributable to the mutant SR Ca ** release channel, and that a wild-type gene encoding the SR Ca ** release channel will have utility in a gene therapy approach for alleviating this disease.
  • transgenic Bio 14.6 hamsters are created that carry and express a wild-type SR Ca ** release channel transgene. If the Bio 14.6 hamster's genetic predisposition to idiopathic dilated cardiomyopathy is due to the absence of a gene encoding the wild-type SR Ca ** release channel, then the presence of the transgene is expected to rescue the animals from developing cardiomyopathy.
  • Example 15 illustrates a procedure which can be used to demonstrate that wild-type gene sequences encoding the SR Ca ** release channel can rescue Bio 14.6 hamsters from developing cardiomyopathy.
  • a transgenic Bio 14.6 hamster that harbored a wild-type SR Ca ** release channel gene remains healthy while age-matched Bio 14.6 animals develop symptoms associated with idiopathic dilated cardiomyopathy.
  • Example 15 Transgenic Bio 14.6 Hamsters Do Not Develop Cardiomyopathy
  • plasmids harboring either the wild-type human or hamster SR Ca ** release channel cDNAs under transcriptional control of a constitutive promoter are first created.
  • the plasmids are constructed such that mRNAs encoding the SR Ca ** release channel proteins can be transcribed from the plasmid-borne promoters when introduced into cardiac myocytes.
  • the plasmids are separately microinjected into oocytes obtained from Bio 14.6 hamsters that are genetically predisposed to develop idiopathic dilated cardiomyopathy.
  • Pups bearing the human or hamster transgenes are bom and identified by standard procedures that will be appreciated by those having ordinary skill in the art, such as through Southern blotting.
  • the transgenic animals are raised to adulthood alongside age-matched Bio 14.6 hamsters that did not receive the transgene.
  • Transgenic hamsters bearing either the hamster or human SR Ca ** release channel transgene are healthy at 400 days of age.
  • Bio 14.6 animals not receiving the transgene exhibit symptoms associated with cardiomyopathy. These symptoms include lethargy and shortness of breath.
  • the animals are sacrificed after breeding and a postmortem examination is conducted.
  • the hearts of the transgenic animals appear substantially identical to hearts isolated from control animals not genetically predisposed to cardiomyopathy.
  • the hearts of-the Bio 14.6 animals appear enlarged to a point nearly double the size of normal control hearts.
  • Transgenic technology can be employed in a second approach for demonstrating that a mutant fo ⁇ n of the SR Ca ** release channel plays a causal role in the development of idiopathic dilated cardiomyopathy.
  • targeted replacement of the two wild-type copies of the gene encoding the SR Ca* * release channel will be carried out to produce transgenic mice.
  • transgenic mice will be obtained that possess two mutant copies of the gene encoding the SR Ca * * release channel.
  • transgenic mouse having both wild-type copies of the SR Ca *+ release channel gene replaced with mutant forms ofthe gene will exhibit disease symptoms.
  • Example 16 describes a procedure that can be used to produce a transgenic mouse that is susceptible to idiopathic dilated cardiomyopathy.
  • a standard "in-out" targeting procedure is used to introduce point mutations into the SR Ca ** release channel gene of a murine embryonic stem (ES) cell line.
  • ES murine embryonic stem
  • One example of such a stem cell line useful for this pu ⁇ ose has been disclosed by Valancius et al. in Mol. Cell. Biol. 11:1402 (1991).
  • the point mutations in the SR Ca ** release channel gene correspond to the mutations which we demonstrated to be characteristic of humans and Bio 14.6 hamsters that are afflicted with idiopathic dilated cardiomyopathy.
  • the vectors are introduced into ES cells by elect roporation.
  • Recombinants are obtained by direct selection in hypoxanthin-aminopterin- thymidine medium according to established procedures known to those having ordinary skill in the art. This targeting procedure allow for easy identification ofthe recombinant cell lines yet does not leave extraneous sequences in either the target or any other region of the genome in the final product.
  • this subtle modification of the SR channel gene could be used to mimic human genetic disease resulting from small specific mutations.
  • transgenic mice homozygous for the mutation in the SR Ca ** release channel gene exhibit a cardiomyopathic phenotype, then such results confirm a cause and effect relationship between the mutant form ofthe SR Ca ** release channel and development of idiopathic dilated cardiomyopathy. Further, a demonstration that substitution of the mutant form of the gene encoding the SR Ca ** release channel for the wild-type form of the gene provides strong evidence that the wild-type form of the gene has utility in gene therapy applications for correcting the genetic defect that causes idiopathic dilated cardiomyopathy.
  • Example 17 describes a gene therapy procedure that can be used to compensate for the genetic defect associated with idiopathic dilated cardiomyopathy in laboratory animals at risk of developing the disease.
  • Example 17 Gene Therapy Applications Based on Expression of the Wild-type SR Ca++ Release Channel - Animal Model
  • a young Bio 14.6 hamster genetically predisposed to develop idiopathic dilated cardiomyopathy is first obtained.
  • a cardiac catheter is positioned within a region of the heart that is to receive a therapeutic gene.
  • a therapeutically effective dose of a composition comprising an expression vector that directs in vivo expression of the wild-type SR Ca* * release channel is then administered to the heart through the catheter. The catheter is withdrawn and the hamster is allowed to recover.
  • the hamster that received the therapeutic composition remains active and healthy at 400 days of age.
  • Age matched Bio 14.6 hamsters that did not receive the therapeutic composition manifest symptoms associated with cardiomyopathy.
  • Example 18 describes a gene therapy procedure that can be used to compensate for the genetic defect associated with idiopathic dilated cardiomyopathy in humans.
  • a cardiac catheter is positioned within a region of the individual's heart that is to receive a therapeutic gene.
  • a therapeutically effective dose of a composition comprising an expression vector that directs in vivo expression of the wild-type SR Ca ** release channel is then administered to the individual's heart through the catheter. The catheter is withdrawn and the individual is allowed to recover. Two weeks following this procedure, a tissue sample containing cardiac myocytes is obtained from the patient. The recovered myocytes are tested and found to express the wild-type SR Ca ** release channel. The individual does not develop idiopathic dilated cardiomyopathy.
  • transgenic mice harboring a mutant SR Ca ** release channel transgene will be useful for studying gene therapy approaches for correcting idiopathic dilated cardiomyopathy.
  • Transgenic hamsters rescued from the idiopathic dilated cardiomyopathy phenotype by a wild type SR Ca ** release channel transgene will be useful for testing drugs that target the multiple gene defects responsible for the disease. Since the transgenic hamster will harbor one fewer genetic defect than the parental Bio 14.6 strain, studying the relative contributions of the remaining gene defects will become more manageable.
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE: (ix) FEATURE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE YES
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE: (ix) FEATURE:

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Abstract

Une mutation génétique au sein du canal de libération de calcium du réticulum sarcoplasmique sert d'indicateur de la prédisposition à la cardiomyopathie idiopathique avec dilatation. On effectue un test qui permet de détecter la présence de la mutation dans un échantillon d'acide nucléique prélevé chez l'individu testé. L'une des techniques que l'on peut utiliser pour ce test est le polymorphisme de taille des fragments de restriction. Une souris transgénique présente un génome contenant au moins un exemplaire d'un polynucléotide codant un canal mutant de libération de Ca++ du réticulum sarcoplasmique. L'un des procédés permettant d'alléger les symptômes associés à la cardiomyopathie idiopathique avec dilatation chez un patient consiste à apporter aux cellules cardiaques du patient un nucléotide codant un canal fonctionnel de libération de Ca++ du réticulum sarcoplasmique.
PCT/US1996/014081 1995-09-19 1996-09-03 Mutation genetique chez les patients atteints de cardiomyopathie idiopathique avec dilatation WO1997011089A1 (fr)

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Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BIOPHYSICAL JOURNAL, 12-16 February 1995, Vol. 68, No. 2, TUNWELL et al., "Expression Studies With cDNAs Encoding the Human Cardiac Ryanodine Receptor-Calcium Release Channel", page A52, Abstract No. M-Pos136. *
CIRCULATION RESEARCH, July 1992, Vol. 71, No. 1, BRILLANTES et al., "Differences in Cardiac Calcium Release Channel (Ryanodine Receptor) Expression in Myocardium From Patients With End-Stage Heart Failure Caused by Ischemic Versus Dilated Cardiomyopathy", pages 18-26. *
CIRCULATION, 15 August 1995, Vol. 92, No. 4, MEYER et al., "Alterations of Sarcoplasmic Reticulum Protein in Failing Human Dilated Cardiomyopathy", pages 778-784. *
CIRCULATION, 15 January 1995, Vol. 91, No. 2, SCHWARTZ et al., "Molecular Basis of Familial Cardiomyopathies", pages 532-540. *
JOURNAL OF BIOLOGICAL CHEMISTRY, 05 February 1990, Vol. 265, No. 4, ZORZATO et al., "Molecular Cloning of cDNA Encoding Human and Rabbit Forms of the Ca2+ Release Channel (Ryanodine Receptor) of Skeletal Muscle Sarcoplasmic Reticulum", pages 2244-2256. *
JOURNAL OF BIOLOGICAL CHEMISTRY, 15 August 1990, Vol. 265, No. 23, OTSU et al., "Molecular Cloning of cDNA Encoding the Ca2+ Release Channel (Ryanodine Receptor) of Rabbit Cardiac Muscle Sarcoplasmic Reticulum", pages 13472-13483. *

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