WO1993017104A1 - Sequence adn du gene de dystrophie myotonique et ses utilisations - Google Patents

Sequence adn du gene de dystrophie myotonique et ses utilisations Download PDF

Info

Publication number
WO1993017104A1
WO1993017104A1 PCT/US1993/001545 US9301545W WO9317104A1 WO 1993017104 A1 WO1993017104 A1 WO 1993017104A1 US 9301545 W US9301545 W US 9301545W WO 9317104 A1 WO9317104 A1 WO 9317104A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
affected
sequence
individual
ctg
Prior art date
Application number
PCT/US1993/001545
Other languages
English (en)
Inventor
J. David Brook
David E. Housman
Original Assignee
Massachusetts Institute Of Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Massachusetts Institute Of Technology filed Critical Massachusetts Institute Of Technology
Publication of WO1993017104A1 publication Critical patent/WO1993017104A1/fr
Priority to US08/422,706 priority Critical patent/US5977333A/en
Priority to US08/422,699 priority patent/US5955265A/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4707—Muscular dystrophy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • DM Myotonic dystrophy
  • DM patients often suffer from cardiac conduction defects, smooth muscle involvement, hypersomnia, cataracts, abnormal glucose response, and, in males, premature balding and testicular atrophy (Harper, P.S., Myotonic Dystrophy. 2nd ed. , W.B. Saunders Co., London, 1989).
  • One of the striking features of this disorder is the variability of phenotype, both within and between families.
  • patients are often subdivided into three groups according to the clinical syndrome and age at onset of the disorder (Harper, P.S. and Dyken, P.R. , Lancet. 2.553-55 (1972)).
  • the mildest form which is occasionally difficult to diagnose, is seen in middle or old age and is characterized by cataract with little or of muscle involvement.
  • the classical form showing myotonia and muscle weakness, most frequently has onset in early adult life and in adolescence.
  • the most severe form which life and in adolescence.
  • the most severe form which occurs congenitally, is associated with generalized muscular hypoplasia, mental retardation, and high neonatal mortality.
  • Those congenitally affected offspring 5 surviving the neonatal period invariably exhibit the classical form of the disease in late childhood or adolescence.
  • the congenital form of DM is almost exclusively maternally transmitted.
  • the phenomenon of anticipation Howeler, C.J. et al.. Brain, 112;779-797
  • Applicants have identified a CTG triplet repeat, present on chromosome 19, which undergoes expansion in myotonic dystrophy (DM) patients. They have also shown that the normal population exhibits great variability in this sequence, which is present in unaffected individuals in 5-27 copies; that DM patients who are minimally af ⁇ fected have at least 50 CTG repeats; and that more severe ⁇ ly affected patients have expansion of the repeat- containing segment up to several kilobase (kb) pairs. In addition, Applicants have demonstrated that the CTG repeat is transcribed and is located in the 3' untranslated region of an RNA which encodes a polypeptide which is a member of the protein kinase family and is expressed in tissues affected by DM.
  • the work described herein makes available a method by which a nucleotide sequence, specifically a CTG triplet repeat, shown to be expanded in individuals affected with DM can be identified in a sample obtained from an indivi- dual.
  • the present method can be used to identify indivi ⁇ duals in whom the CTG triplet repeat is present in normal copy number and individuals in whom the CTG triplet repeat occurs in abnormally high copy number, as well as to further identify individuals likely to be minimally affected and individuals likely to be more severely affected.
  • RNA which includes in its 3' untranslated region the transcribed CTG triplet repeat, encodes a protein kinase. It is reasonable to expect that amplification of the CTG triplet repeat affects the function of the DM gene, such as by causing a loss of expression of the allele carrying the expanded repeat or by causing a gain of function in the DM gene (e.g., deletion or inactivation of a binding site for a negative control element) .
  • the protein kinase encoded by the DM gene can be used as the basis for a method of identifying individuals affected by DM, since the mRNA is expressed in tissues affected by DM.
  • the presence or absence, as well as the level of expression, of the protein kinase can be determined in tissues using, for example, polyclonal or monoclonal antibodies which recognize (bind) the protein kinase.
  • the DM gene or mRNA can be detected and/or quantitated using DNA probes described herein and artrecognized hybridization techniques.
  • Figure 1 is a restriction map of the 10 Kb EcoRI fragment that undergoes expansion in myotonic dystrophy patients. Restriction sites for BamHI(B), EcoRI(E), Hindi(C), Hind ⁇ II(H), Pst ⁇ (P) and Saci(S) are indicated. The subcloned 1.4 Kb Ba HI fragment (pMIOM-6) is shown enlarged with the PstI and the Hindi sites, which flank the expanded region. The positions of PCR primers 96, 98, 101, 102 and 103 and the sequence between primers 101 and 102 (SEQ ID No. 1 and SEQ ID No. 2 respectively) are shown.
  • Figure 2 shows results of Southern blot analysis of the DNA fragment which is expanded in DM.
  • Figure 4 is the pedigree of a myotonic dystrophy family with their genotypes at the DM locus.
  • Figure 5 is the nucleotide (SEQ ID NO: 3) and the deduced amino acid sequence (SEQ ID NO: 4) of cDNA C28.
  • Figure 6 is the nucleotide sequence of the DM gene, (SEQ ID NO: 13) including the deduced amino acid sequences of the three predicted reading frames (sequence a, upper line, SEQ ID NO:14; sequence b, middle line, SEQ ID NO:15; sequence c, lower line, SEQ ID NO:16) .
  • sequences of the intron/exon boundaries have been determined and are indicated by the XXX sites located within the sequence (the actual sequences are not shown) .
  • Figure 7 is a graphic representation of the alternate splice sites of the DM gene and of the resulting cDNAs.
  • RNA which encodes a polypeptide with strong amino acid homology to members of the protein kinase gene family and is expressed in many of the tissues affected in DM.
  • the modal number of CTG triplet repeats in 282 unaffected individuals tested has been shown to be 5, with the largest number being 27.
  • individuals minimally affected by DM have been shown to have at least 50 CTG triplet repeats and more severely affected individuals have been shown to have even greater numbers of copies (e.g., expansion of the repeat- containing sequence by as much as several Kb pairs) .
  • the protein kinase-like polypeptide has been shown to be highly expressed in heart, expressed to a lesser extent in muscle and also expressed in brain.
  • a method of determining in DNA obtained from an individual, the copy number of the CTG triplet repeat which has been shown to be increased or expanded in DNA individuals affected with muscular dystrophy is available.
  • a method of determining whether an individual is likely to be affected with myotonic dystrophy is also available.
  • DNA is obtained from an individual to be assessed and the copy number of CTG triplet repeats on chromosome 19 DNA is determined. If the copy number of CTG triplet repeats is at least 50, the individual is likely to be affected with myotonic dystrophy.
  • DNA can be obtained from a variety of tissues (e.g., blood, muscle, skin) , either prenatally or postnatally.
  • the present work also provides a method of deter ⁇ mining whether an individual is likely to be affected with myotonic dystrophy in which the expression of the protein kinase encoded by the DNA of Figure 5 or Figure 6 (or a protein kinase having a substantially equivalent sequence) is detected, particularly, in tissues affected by myotonic dystrophy (e.g., heart, brain and muscle).
  • a tissue sample to be analyzed is obtained from an individual to be assessed for the likelihood he or she will be or is affected with myotonic dystrophy.
  • expression of the protein kinase can be determined through the use of an antibody specific for (one which binds) the protein kinase described herein.
  • the antibody used can be polyclonal or monoclonal and is contacted with the tissue to be assessed, after the tissue has been processed or treated to render the protein kinase (if present) available for binding by the antibody. Binding of the antibody to a component of the tissue sample is indicative of the presence of the protein kinase and, thus, of the likelihood the individual will be or is affected with myotonic dystrophy.
  • the work described herein also makes available antibodies specific for (which bind to) the protein kinase encoded by the DNA sequence of Figure 5 or Figure 6 or an equivalent protein kinase (a protein kinase encoded by a substantially similar DNA sequence and/or having substantially the same amino acid sequence as that represented in Figure 5 or Figure 6) .
  • a protein kinase is expressed in tissues affected by myotonic dystrophy.
  • the effects of the protein kinase can be reduced (totally or partially) by administering to an individual affected with or likely to be affected with myotonic dystrophy a drug which interferes with the kinase activity, either directly or indirectly.
  • a drug which inter ⁇ feres with expression of the protein kinase e.g., a nucleotide sequence which binds to the kinase-encoding sequence and prevents it from being transcribed/expressed
  • a drug which inter ⁇ feres with expression of the protein kinase e.g., a nucleotide sequence which binds to the kinase-encoding sequence and prevents it from being transcribed/expressed
  • mapping of the DM region of chromosome 19, which is increased in size in DM is increased in size in DM
  • identification of the CTG triplet repeats on chromosome 19 and assessment of the number of repeats in normal and affected individuals identification and characterization of genomic clones spanning the 10 kb fragment which is increased in size in DM
  • the cDNAs which were isolated from various libraries by probing with one of the genomic clones and the full length DNA sequence of the DM gene.
  • Probes pBB0.7 (Harley, H.G. et al.. Nature 355:545-546 (1992) and cDNA 25 (Buxton, J. et al.. Nature 355:547-548 (1992) identify the same EcoRI restriction fragment length polymorphism in the normal population, with alleles of 9 kb or 10 kb. In 43 of 53 unrelated affected individuals reported in these two studies, only one of the normal-sized alleles is present, plus an additional, larger, disease-specific band. This restric- tion fragment varies in length between patients, even between siblings within the same family.
  • variable fragment increases in successive generations and shows a correlation between increased severity and earlier onset of the disease.
  • the largest fragment detected is 15 kb, an increase of 5 kb over the normal size (Harley, H.G. et al.. Nature 355:545-546 (1992) .
  • genomic clones spanning the 10 kb fragment that is increased in size in this disease were characterized.
  • the modal number of repeats found in 282 normal alleles surveyed is 5 (48%) , with the largest being 27.
  • Minimally affected DM patients have at least 50 copies.
  • the CTG repeat is transcribed and is found at a position 500 bp from the poly(a) tract of an mRNA expressed in many of the tissues affected in DM.
  • the RNA in which the repeat resides encodes a polypeptide with strong amino acid homology to members of the protein kinase gene family.
  • FIG. 1 shows a detailed restriction map of this interval. Sites for BamHI, Hindi, Hindlll, PstI, and Sad are marked. Single-copy probes mapping within this EcoRI fragment were hybridized to DNA from patient and normal individuals digested with a series of restriction enzymes. Figure 2 shows the result of one such hybridization. The normal control is heterozygous for an EcoRI RFLP.
  • Amplifi ⁇ cation by PCR between oligos 98 and 100 produced single bands that were identical in size in patient, normal and pM10M-6 lanes when visualized on ethidium-stained agarose gels.
  • PCR using oligos 96 and 103 produced two bands in a normal human sample, a single band in DM DNA and a single band in pM10M-6 that was smaller than any of the other bands.
  • Analysis of the sequence derived from pM10M-6 between oligos 96 and 103 revealed tandem repeats of the trinucleotide CTG. Two other oligos, 101 and 102, which more closely flank this triplet repeat, were tested using PCR. These produced similar but more striking band size differences than with oligos 96 and 103 because of the smaller PCR product.
  • the sequence of pM10M-6 between oligos 101 and 102 is shown in Figure 1.
  • PCR products analyzed on ethidium-stained gels showed that one individual from each pair had two alleles in the normal range, whereas the other grandparent (in each case, the one with cataracts) had one band in the normal range in addition to a second diffuse band at about 250 bp.
  • For accurate sizing of this larger band labeled PCR products were analyzed on sequencing gels. Although unrelated, these two individuals had very similar larger bands (corresponding to 50-55 repeats) .
  • the larger allele was amplified using PCR and cloned and sequenced, in order to determine whether the increased fragment size in these patients is due entirely to the expansion of the CTG repeat.
  • a DM family that shows increased severity of disease in successive generations is shown in Figure 4.
  • a band of approximately 300 bp is observed in the PCR product of four family members in the first two generations shown in the pedigree. This result demonstrates that a CTG repeat unit of approximately 60 repeats can be transmitted from one generation to the next without an obligatory expan ⁇ sion.
  • Individuals in the first two generations had mild symptoms such as cataracts or were apparently asympto ⁇ matic.
  • individuals in the third and fourth generations of the family showed severe symptoms of DM. These individuals showed only a single PCR band within the normal range. Southern blotting analysis confirmed that, for these individuals, as for most other severely affected DM patients, a dramatic expansion in allele size has occurred to increase the repeat size beyond that which can be detected by PCR analysis.
  • the triplet repeat sequences amplified in fragile-X syndrome and X-linked spinal and bulbar muscular atrophy (SBMA) are expressed in mRNAs.
  • SBMA X-linked spinal and bulbar muscular atrophy
  • clone pM10M-6 was hybridized to a Southern blot of DNA from different species. Results showed that DNA sequences contained within this clone are strongly conserved, suggestive of a transcribed sequence.
  • pM10M-6 was used to screen cDNA libraries derived from several different sources, including adult frontal cortex, substantia nigra, fetal muscle and fetal brain. A total of 10 6 clones were screened from these libraries and 110 positives were identified.
  • C28, C31, C34, C35, C39 and C85 The insert size of each clone and the library from which it was derived are: C28 (frontal cortex), 2.5 kb; C31 (frontal cortex), 2.1 kb; C34 (substantia nigra), 1.7 kb; C35 (fetal brain), 1.7 kb; C39 (frontal cortex), 2.7 kb; C85 (fetal muscle), 2.8 kb. All six clones were hybri ⁇ dized to a panel of hybrid cell lines to confirm that they were from the expected region of chromosome 19.
  • cDNAs C28 and C85 each span at least 10 kb of genomic DNA. Clone C39 was chimeric at the 5' end. whereas the others mapped as expected. Clones C28, C34 and C35 were completely sequenced and clone C85 was partially sequenced. All clones contained the CTG repeat, and this varied in length between clones. Clones C28, C34, C35, and C85 contained 11, 5, 12, and 13 triplets, respectively.
  • the C28 sequence was compared to the nonredundant sequence database, which combines all avail ⁇ able protein databases.
  • This sequence search revealed homology to the cyclic AMP-dependent protein kinases. The highest score was to the protein kinase TKR-YKR from Saccharomyces cerevisiae. The 11 protein kinase domains are found within the first 300 residues of deduced amino acid sequence. Beyond the kinase domain, some slight homology to the chicken myosin heavy chain was observed. The disease, myotonic dystrophy shows a clear autosomal dominant pattern of inheritance. However, there are several aspects of this disorder that are particularly challenging to explain from a molecular genetic perspective.
  • DM and fragile-X are categorized as normal premutation or full mutation on the basis of CGG repeat number (Fu, Y.-M et al.. Cell 57:1047- 1058 (1991)) . Transmissions of 46 repeats and below are within the normal range and are stable. The transition from stability to instability occurs within the 46-52 repeat range. Premutations showing no phenotypic effect range from 52 to 200 repeats and are meiotically unstable. For myotonic dystrophy there is, as yet, no clear classi ⁇ fication of premutation. However, it seems likely that minimally affected individuals could go undiagnosed, were it not for the appearance of a more severely affected individual in a subsequent generation.
  • the DM gene must indeed be extremely sensitive to gene dosage, since gene expression levels in the presence of a normal allele can range only between 50% and 100% of normal. Genomic imprinting cannot be invoked to increase this range too much further, since DM can be inherited from either the father or the mother, with quite severe symptoms.
  • the expansion of the CTG repeat may lead to a gain of function in the DM gene.
  • Gain-offunction mutations in the 3' UTR of the fem-3 and lin-14 genes of Caenorhabditis ele ⁇ ans have recently been demonstrated (Ahringer, J. and J. Kimble, 5 Nature 349:346-348 (1991); .Wightman, B. et al.. Gene Dev. 1:1813-1824 (1991)). In both cases, deletion or inactivation of a binding site for a negative control element is thought to result in unregulated activity of these genes. Amplification of the CTG repeat in DM may be
  • cAPK cAMP-dependent protein kinase
  • PCR analysis was performed as follows. Reactions (10 ⁇ l) were set up using standard PCR conditions (50 mM KC1, 1.5 mM MgCl 2 , 10 mM Tris [pH 8.3], 200 //M dNTPs, 1 ⁇ VL of each primer, and 20 ng template DNA) .
  • oligo 101 was incubated for 30 min at 37 ⁇ C with T4 polynucleotide kinase (3 ⁇ ) in 20 ⁇ l reaction with 50 mM Tris-HCl (pH 7.5).
  • PCR reaction was spiked with the labeled primer in a ratio of 30:1 unlabeled to labeled.
  • PCR reactions were carried out on a Stratagene 96 well thermocycler. Cycling conditions were as follows: 1 x (3 min, 94 * C) , 35 x (10s, 94'C; 30s, 62 ° C; 30s, 72 * C) , 1 x (5 min, 72°C) , 15°C soak. Samples then were either loaded on agarose gels or were diluted 1:1 with sequencing buffer, denatured and loaded on 8% sequencing gels.
  • sequences of oligos used in PCR reactions was as follows: 96, GGT GCG TGG AGG ATG GAA CAC GGA C (SEQ ID NO: 5) 98, GCG TGC GAG TGG ACT AAC AAC AGC TG (SEQ ID NO: 6)
  • Genomic DNAs were digested with restriction endonuc- leases PstI Sad, EcoRI or BamHI (New England Biolabs)* in 30 ⁇ l or 40 ⁇ l reactions with NE Buffer 10 x according to the manufacturer's instructions. Digested DNAs were run on 0.8% agarose gels (FMC), denatured in 0.5 M NaOH, 1.5 M NaCl, neutralized in 1 M Tris (ph 7.0), 1.5 M NaCl and transferred to zetabind (AMF) membranes in 10 x SSC.
  • FMC 0.8% agarose gels
  • AMF zetabind
  • Hybridizations to both Northern and Southern blots were performed at 42 ⁇ C in 50% formamide with 5 x SSC, 1 x Denhardt's solution, 0.02 M NaP0 4 , 100 ⁇ g/ml single- stranded DNA, 10% dextran sulphate. DNA probes were labeled by random priming (Feinberg, A.P. and B.
  • DNA databases were searched on a digital VAX computer using the GCC software package (Altschul, S.F. et al.. J. Mol. Biol. 215:403-410 (1990)) and the BLAST network service from the National Center for Biotechnology Information.
  • One hybrid, 2F5 contains 2-3 megabases of human material, derived exclusively from human chromosome 19 and includes markers which flank DM. DNA from this hybrid was used to construct genomic phage libraries from which 230 phage containing human inserts have been identified. Two other hybrids produced provide breakpoints within the interval covered by 2F5 and are useful in subdividing the phage clones into three groups.
  • Cell line 20XP3542-1-4 was used as the parental cell line in two different X-irradiation experiments.
  • Cell line DNAs were analyzed with twenty-three DNA markers which were either positive in or derived from parental cell line 20XP3542-1-4. These were divided into two series.
  • Series 1 DNA markers included BCL3, AP0 ⁇ c2, CKM, ERCC1, NE16, pD26 (D17S243) and pD48 (D8S42) .
  • Series 2 DNA markers include pD3 (D19S61) , pD8 (D19S62), pDIO (D19S63) , pD36 (D19S64) , pNE17, pD50
  • a genomic DNA library was constructed from cell line 2F5 in vector Lambda DASH (Stratagene) by partial Mbol digestion of cell line DNA, size selection of 15-25 Kb fragments on low melting point agarose gels and cloning into the BamHI site of the vector. Recombinants were plated on bacterial strain NM542 and screened with total human DNA. Southern blots were prepared from DNA of 35 phage digested with restriction enzymes BamHI, EcoRI, Hindlll and Sail and hybridized with human DNA. Those bands not hybridizing well with human DNA were identified and excised from LMP agarose gel containing digests of the same phage and hybridized against mapping filters.
  • PK-87-19 contains a single chromosome 19 as its only human chromosome and GM89A99c7 contains the region 19ql3"3-19qter plus chromo ⁇ somes 3, 4, 7, 11, 18, 21, 22 and Xpter-Xq24.
  • Phage lambda libraries were constructed from DNA fractionated by preparative PFGE. Approximately 100 ⁇ g (16 blocks) of 2F5 hybrid DNA was digested .with Notl and separated by PFGE. After electrophoresis, the outside lanes containing size markers were cut off and stained with ethidium bromide. The gel was re-assembled and the central section containing the fractionated hybrid cell line DNA, was cut into 2 mm slices at right angles to the direction of electrophoresis. These were melted at 65°C, cooled to 37°C and the agarose was removed by digestion with agarose followed by phenol and chloroform extraction.
  • the DNA was recovered by ethanol precipitation, a small aliquot of each fraction was digested with PstI, the samples were separated by standard gel electrophoresis, blotted and hybridized with various probes to determine in which fractions the corresponding Notl fragments were present.
  • DNA from the chosen fractions was then partially digested with Mbol to 15-25 kb average size. Due to the small amount of DNA available, the partial digest con ⁇ ditions were established by electrophoresis of the trial samples in 0.6% agarose gels, followed by blotting and hybridization with labelled Chinese hamster DNA.
  • the partial digests were cloned in two ways: firstly, using lambda EMBL3 cut with BamHI in order to obtain Mbol fragments internal to the original Notl fragment; and secondly, with a derivative of lambda EMBL3 in which one of the BamHI cloning sites was replaced with a Notl site. This allowed the ends of the Notl fragment to be obtained.
  • the ligated DNAs were packaged in vitro and plated on IJ . coli strain ER1458. Phage with human inserts were identified by hybridization with labelled total human DNA.
  • DNA from 2F5 cells was prepared in agarose plugs for use as PCR template (van Oremen, G.J.B. and Verkerck, In: Human Genetic Diseases , A Practical Approach IRL Press, Oxford (1986)).
  • PCR primers Alu-1 and Alu-22 that specifically recognize human consensus sequences located at the 5' and 3' ends of Alu segments, were used together with 2F5 template to amplify human unique sequences (Liu, et al.. submitted) .
  • Alu-1 and Alu-2 sequences were GGATTACAGGYRTGAGCCA (SEQ ID NO: 11) " and
  • RCCAYTGCACTCCAGCCTG SEQ ID NO: 12 respectively, where Y is either pyrimidine (T or C) and R is either purine (A or G).
  • l ⁇ g of PCR product was labelled with biotin-7-dATP using a nick translation kit (BRL cat. no. 8160SB) . Free nucleotides were removed by passing the mixture through a Worthington Sephadex column. The procedure of Pinkel, et al. was followed for in situ hybridization with modifi- cations described in Doll, et al. (Pinkel, D. et al.. Proc. Natl. Acad. Sci. USA 83:2934-2938 (1986); Doll, G. et al..
  • Cell lines from each of the radiation treatment groups were analyzed with two different sets of DNA markers. Three cell lines in particular appeared very useful and formed the basis of further analyses.
  • Cell line 2F5 had lost all the non-chromosome 19 derived markers present in the parent cell line 20XP3542-1-4. Furthermore, it had also lost the four most proximal markers from chromosome 19; PVS, BCL2, AP0C2 and CKM, while retaining the other chromosome 19 markers including ERCC1 and pl34C (D19S51) which flank DM.
  • Hybrid line 5B3 retained even fewer markers than 2F5, however, non-contiguous pieces of chromosome 19 were present in this case.
  • CKM which maps between APOC2 and ERCCl
  • CKM which maps between APOC2 and ERCCl
  • Marker pl34C (D19S51)
  • the closest marker flanking DM on the distal side was also deleted from 5B3. Nevertheless, this cell line was useful for subdividing the region of chromosome 19 distal to ERCCl.
  • cell line 3A3 also provides a break ⁇ point within this interval. 3A3 had lost several of the distal chromosome 19 markers present in cell line 2F5, while retaining pl34C (D19S51) and other more proximal chromosome 19 markers, as well as several of the non- chromosome 19 markers from the parent cell line.
  • cell lines 3A3 and 5B3 provided a means of assigning DNA clones derived from cell line 2F5 into three intervals. Phage clones present in both 3A3 and 5B3 were assigned to interval 'A'. Those present in 3A3, but absent from 5B3, were assigned to 'B' and those absent from both 3A3 and 5B3 were assigned to interval 'C.
  • DNA from cell line 2F5 was labelled and used as probe on mitotic spreads of human chromosomes. This hybridized to a single region from the long arm of chromosome 19.
  • the human DNA content was also characterized by PFGE.
  • DNA from the hybrids 2F5 and 20XP3542-1-4 was digested with Notl, Mlul and BssHII and separated by PFGE. A blot of the gel was hybridized with total human DNA. 2F5 has a considerably reduced human DNA content compared to its parent cell line. In the Notl digest, fragments hybridizing with human DNA of approximately 50, 180, 200, 400, 500, 1000 and 1300 kb were present.
  • the other two libraries were constructed from PFGE fractionated DNA as described above.
  • the marker D19S63 showed marked linkage disequilibrium and no recombination
  • HTF island subclones Five of the HTF island subclones (p20.1, p36.1, 037.1, p42.3 and p56.1) gave unambiguous localizations on the PFGE map, and mapped to interval 'A' as defined above. Two of these clones (p20.1 and p36.1) were not on the same Notl fragment as D19S63. It is possible that the original Notl digest used in the library construction was .incom ⁇ plete, resulting in a contaminating 250 kb Notl fragment that was not completely resolved from the 200 kb fragment by the preparative PFGE. All of the HTF island subclones detected sequence conservation by zoo-blot analysis and were used to screen cDNA libraries.
  • cell line 20XP3542-1-4 (Stallings et al.. Am. J. Hum. Genet. 41:144-153 (1988)) which contains a single human element 20-30 megabases in size derived from at least four differ ⁇ ent chromosomes including a small part of 19q was used.
  • group l in which the parental cell line was lethally irradiated and fused to DNA repair deficient cell line UV20 followed by selection of clones in mitomycin C and polio virus, produced the most useful clones, in particular 2F5.
  • Hybrid 3A3 resulted from the exposure of the parental cell line to lethal dose irradiation followed by fusion to HPRT deficient hamster cells. As described by Cox et al. (1989) , no selection was employed for the region of interest.
  • the hybrid 2F5 provided a source of DNA specific for the region of chromosome 19 distal to ERCCl.
  • sequence c is the amino acid sequence resulting from the correct predicted open reading frame (the M indicated by the arrow on sequence c indicates the predicted start of the correct reading frame) . Also indicated on Figure 6 (lines and arrows) are the locations of two cDNAs comprising the gene sequence, cDNA 41 and cDNA 28.
  • cDNA 28 isolated form an adult brain library
  • cDNA 35 from fetal brain
  • 41 from fetal muscle
  • both cDNA 35 and 41 contain 15 bases at the 5' end of exon 9 which are not present in cDNA 28.
  • cDNA 41 does not conatin exon 14.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Biomedical Technology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé permettant d'identifier dans un échantillon pris chez un individu une séquence nucléotidique, en particulier une répétition de triplet CTG qui se développe chez des individus atteints de dystrophie myotonique. Le procédé de l'invention peut être utilisé pour identifier des individus chez lesquels la répétition de triplets CTG est présente avec un nombre de reproductions normal et d'identifier des individus chez lesquels la répétition de triplets CTG est présente en un nombre de reproductions anormalement élevé, et également d'identifier des individus susceptibles d'être légèrement affectés et des individus susceptibles d'être plus sévèrement affectés. L'invention a permis d'obtenir une unité ou gène de transcription de la distrophie myotonique et pouvant jouer un rôle important dans la pathophysiologie de la dystrophie myotonique.
PCT/US1993/001545 1992-02-06 1993-02-19 Sequence adn du gene de dystrophie myotonique et ses utilisations WO1993017104A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US08/422,706 US5977333A (en) 1992-02-06 1995-04-14 DNA sequence encoding the myotonic dystrophy gene and uses thereof
US08/422,699 US5955265A (en) 1992-02-06 1995-04-14 DNA sequence encoding the myotonic dystrophy gene and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US83925592A 1992-02-20 1992-02-20
US839,255 1992-02-20

Publications (1)

Publication Number Publication Date
WO1993017104A1 true WO1993017104A1 (fr) 1993-09-02

Family

ID=25279249

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/001545 WO1993017104A1 (fr) 1992-02-06 1993-02-19 Sequence adn du gene de dystrophie myotonique et ses utilisations

Country Status (1)

Country Link
WO (1) WO1993017104A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2718753A1 (fr) * 1994-04-15 1995-10-20 Pasteur Institut Procédé de comptage de mono-, di- et trinucléotides répétés dans un génome eucaryote et trousse permettant la mise en Óoeuvre de ce procédé.
WO1998056950A1 (fr) * 1997-06-11 1998-12-17 Fondation Jean Dausset-Ceph Sequences d'adn riches en triplet repete utiles dans le diagnostic de maladies a repetition trinucleotidique
US5962332A (en) * 1994-03-17 1999-10-05 University Of Massachusetts Detection of trinucleotide repeats by in situ hybridization
WO2010115033A3 (fr) * 2009-04-02 2011-01-27 Regents Of The University Of Minnesota Polypeptides associés à des expansions de répétitions nucléotidiques et leurs utilisations

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CELL. vol. 68, no. 4, 21 February 1992, CAMBRIDGE, NA US pages 799 - 808 BROOK, J.D. ET AL. 'Molecular basis of Myotonic Dystrophy.: Expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member' *
NATURE. vol. 250, 19 July 1974, LONDON GB pages 245 - 247 ROSES, A. D. & APPEL, S. H. 'Muscle membrane protein kinase in myotonic muscular dystrophy' *
NATURE. vol. 355, no. 6360, 6 February 1992, LONDON GB pages 545 - 546 HARLEY, H. G. ET AL. 'Expansion of an unstable DNA region and phenotypic variation in myotonic dystrophy' *
NATURE. vol. 355, no. 6360, 6 February 1992, LONDON GB pages 547 - 548 BUXTON, J. ET AL. 'Detection of an unstable fragment of DNA specific with myotonic dystrophy' *
NATURE. vol. 355, no. 6360, 6 February 1992, LONDON GB pages 548 - 549 ASLSNIDIS, C. ET AL. 'Cloning of the essential myotonic dystrophy region and mapping of the putative defect' *
SCIENCE vol. 255, no. 5049, 6 March 1992, LANCASTER, PA US pages 1256 - 1258 FU, Y. H. ET AL. 'An unstable triplet repeat in a gene related to Myotonic Muscular Dystrophy' *
SCIENCE vol. 256, no. 5058, 8 May 1992, LANCASTER, PA US pages 784 - 788 CASKEY, C. T. ET AL. 'Triplet repeat mutations in human disease' *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5962332A (en) * 1994-03-17 1999-10-05 University Of Massachusetts Detection of trinucleotide repeats by in situ hybridization
FR2718753A1 (fr) * 1994-04-15 1995-10-20 Pasteur Institut Procédé de comptage de mono-, di- et trinucléotides répétés dans un génome eucaryote et trousse permettant la mise en Óoeuvre de ce procédé.
WO1998056950A1 (fr) * 1997-06-11 1998-12-17 Fondation Jean Dausset-Ceph Sequences d'adn riches en triplet repete utiles dans le diagnostic de maladies a repetition trinucleotidique
FR2764611A1 (fr) * 1997-06-11 1998-12-18 Fondation Jean Dausset Ceph Sequences d'adn riches en triplet repete utiles dans le diagnostic de maladies a repetition trinucleotidique
WO2010115033A3 (fr) * 2009-04-02 2011-01-27 Regents Of The University Of Minnesota Polypeptides associés à des expansions de répétitions nucléotidiques et leurs utilisations

Similar Documents

Publication Publication Date Title
Brook et al. Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member
EP0238329B1 (fr) Sondes génétiques
Kwiatkowski et al. Construction of a GT polymorphism map of human 9q
van Geel et al. Genomic analysis of human chromosome 10q and 4q telomeres suggests a common origin
DiLella et al. An ammo-acid substitution involved in phenylketonuria is in linkage disequilibrium with DNA haplotype 2
Wong et al. Characterization of a panel of highly variable minisatellites cloned from human DNA
EP1520039B1 (fr) Polymorphismes mitochondriaux associes a une predisposition au developpement de fibrose ou de cicatrisation inappropriee
US6201107B1 (en) Cystic fibrosis gene
US6429011B1 (en) Neuronal apoptosis inhibitor protein gene sequence and mutations causative of spinal muscular atrophy
US5879884A (en) Diagnosis of depression by linkage of a polymorphic marker to a segment of chromosome 19P13 bordered by D19S247 and D19S394
Ozcelik et al. Synaptophysin: structure of the human gene and assignment to the X chromosome in man and mouse.
Montermini et al. The Friedreich ataxia critical region spans a 150-kb interval on chromosome 9q13
Nakabayashi et al. Molecular genetic studies of human chromosome 7 in Russell–Silver syndrome
JPH07143884A (ja) 腫瘍サプレッサー遺伝子メルリンおよびその用途
Rogner et al. A YAC clone map spanning 7.5 megabases of human chromosome band Xq28
AU744157B2 (en) Novel gene encoding a DNA repair endonuclease and methods of use thereof
US5955265A (en) DNA sequence encoding the myotonic dystrophy gene and uses thereof
JPH05211897A (ja) ヌクレオチド配列
WO1993017104A1 (fr) Sequence adn du gene de dystrophie myotonique et ses utilisations
Cotran et al. Genetic analysis of patients with retinitis pigmentosa using a cloned cDNA probe for the human gamma subunit of cyclic GMP phosphodiesterase
US5556786A (en) Anhidrotic ectodermal dysplasia gene and method of detecting same
JP4637982B2 (ja) ８型脊髄小脳性運動失調及び検査方法
US5977333A (en) DNA sequence encoding the myotonic dystrophy gene and uses thereof
US5830661A (en) Diagnosis and treatment of glaucoma
Zuo Cloning and characterization of the Huntington disease region on human chromosome 4

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA