[go: up one dir, main page]

WO1992000386A1 - Procedes de detection d'atrophie musculaire spinale de type i et de types ii/iii et marqueur a cet effet - Google Patents

Procedes de detection d'atrophie musculaire spinale de type i et de types ii/iii et marqueur a cet effet Download PDF

Info

Publication number
WO1992000386A1
WO1992000386A1 PCT/US1991/004605 US9104605W WO9200386A1 WO 1992000386 A1 WO1992000386 A1 WO 1992000386A1 US 9104605 W US9104605 W US 9104605W WO 9200386 A1 WO9200386 A1 WO 9200386A1
Authority
WO
WIPO (PCT)
Prior art keywords
marker
subject
locus
muscular atrophy
type
Prior art date
Application number
PCT/US1991/004605
Other languages
English (en)
Inventor
Conrad Gilliam
Original Assignee
The Trustees Of Columbia University In The City Of New York
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 The Trustees Of Columbia University In The City Of New York filed Critical The Trustees Of Columbia University In The City Of New York
Publication of WO1992000386A1 publication Critical patent/WO1992000386A1/fr

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
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • 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
    • C12Q2600/112—Disease subtyping, staging or classification
    • 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

  • SM ⁇ Spinal muscular atrophy
  • the childhood-onset SMAs describe a heterogeneous group of disorders that rank second in frequency to cystic fibrosis among autosomal recessive disorders and are the leading cause of heritable infant mortality.
  • Acute SM ⁇ (SM ⁇ Type I/Werdnig- Hoffmann/severe/infantile) is the leading cause of heritable infant mortality.
  • the subject invention discloses that the locus for Type I SM ⁇ is homologous with the locus for Type II/III SMA. i.e. that Type I and Type II/III SM ⁇ map to the same locus.
  • This invention also discloses that the SM ⁇ locus is in an 11 centimorgan region within the 5qll.2-l3.2 region of chromosome 5.
  • This invention further discloses markers for Type I and Type II/III SM ⁇ , in particular the marker D5S112. Melki, et al. (30) demonstrate the linkage of a marker, D5S39, to Type II/III SM ⁇ .
  • SM ⁇ has heretofore been diagnosed by use of phenotypic tests such as electromyographs, and by muscle biopsy. Such tests do not detect SMA carriers, and are impracticable for prenatal diagnosis.
  • the subject invention represents a genetic test for the diagnosis of SM ⁇ . It can be used to detect adult carriers, and can be used prenatally to detect affected fetuses. The high incidence of this disease makes the efficient diagnosis provided by this test a public health priority.
  • This invention provides a method for determining whether a subject is likely to develop Type I spinal muscular atrophy by detecting a mutation at the Type I spinal muscular atrophy, locus, which comprises a. isolating DNA from the subject, b. digesting the subject's isolated DNA with at least one restriction enzyme for a marker mapping to the locus 5qll.2-13.3 in close proximity to the Type I spinal muscular atrophy locus and which detects a restriction fragment length polymorphism with this enzyme which is consistently associated with a mutation at the Type I spinal muscular atrophy locus, c. electrophoresing the subject's resulting DNA fragments on a gel, d. attaching a detectable label to at least one marker mapping to the locus 5qll.2-
  • This invention also provides a method for determining whether a subject is likely to develop Type II/III spinal muscular atrophy by detecting a mutation at the Type II/III spinal muscular atrophy locus, which comprises, a. isolating DNA from the subject, b. digesting the subject's isolated DNA with at least one restriction enzyme for a marker mapping to the locus 5qll.2-13.3 in close proximity to the Type II/III spinal muscular atrophy locus and which detects a restriction fragment length polymorphism consistently associated with a mutation at the Type II/III spinal muscular atrophy locus, c. electrophoresing the subject's resulting DNA fragments on a gel, d.
  • a detectable label to at least one marker mapping to the locus 5qll.2- 13.3 which has a restriction fragment length polymorphism for a restriction enzyme of step b consistently associated with a mutation at the Type II/III spinal muscular atrophy locus, e. contacting the subject's DNA fragments of step c with at least one detectably labelled marker of step d under conditions such that the detectably labelled marker hybridizes to the appropriate DNA fragments, f. visualizing the resulting detectably labeled DNA fragments, and g.
  • This invention further provides a method for determining whether a subject is likely to develop Type I spinal muscular atrophy by detecting a mutation at the Type I spinal muscular atrophy locus, which comprises, a. isolating DNA from the subject, from a normal member of the subject's family, and from at least one member of the subject's family who suffers from Type I spinal muscular atrophy, b. digesting the isolated DNAs with at least one restriction enzyme for a marker mapping to the locus 5qll.2-l3.3 in close proximity to the Type I spinal muscular atrophy locus ⁇ and which detects a restriction fragment length polymorphism consistently associated with a mutation at the Type I spinal muscular atrophy locus, c. electrophoresing the resulting DNA fragments on a gel, d.
  • a detectable label to at least one marker mapping to the locus 5qll.2-13.3 which has a -restriction fragment length polymorphism for a restriction enzyme of step b consistently associated with a mutation at the Type I spinal muscular atrophy locus, e. contacting the DNA fragments of step c with at least one detectably labelled marker of step d under conditions such that the detectably labelled marker hybridizes to the appropriate DNA fragments, f. visualizing the resulting detectably labeled DNA fragments, g.
  • determining by the number and size of the subject's DNA fragments which have hybridized to the marker whether the subject's DNA displays a pattern of restriction fragment length polymorphisms consistently associated with a mutation at the Type I spinal muscular atrophy by determining whether the hybridized fragments resulting from the DNA isolated from the subject are the same in number and size as the hybridized fragments resulting from the DNA isolated from the normal member of the subject's family, or the hybridized fragments resulting from the DNA isolated from the member of the subject's family who suffers from Type I spinal muscular atrophy, thereby detecting a mutation at the Type I spinal muscular atrophy locus.
  • This invention also provides a method for determining whether a subject is likely to develop Type II/III spinal muscular atrophy by detecting a mutation at the Type II/III spinal muscular atrophy locus, which comprises, a. isolating DNA from the subject, from a normal member of the subject's family, and from at least one member of the subject's family who suffers from Type II/III spinal muscular atrophy, b. digesting the isolated DNAs with at least one restriction enzyme for a marker mapping to the locus 5qll.2-l3.3 in close proximity to the Type II/III spinal muscular atrophy locus and which detects a restriction fragment length polymorphism consistently associated with a mutation at the Type II/III spinal muscular atrophy locus, c.
  • step d attaching a detectable label to at least one marker mapping to the locus 5qll.2-13.3 which has a restriction fragment length polymorphism for a restriction enzyme of step b consistently associated with a mutation at the Type II/III spinal muscular atrophy locus, e. contacting the DNA fragments of step c with at least one detectably labelled marke of step d under conditions such that the detectably labelled marker hybridizes to the appropriate DNA fragments, f. visualizing the resulting detectably labeled DNA fragments, g.
  • determining by the number and size of the subject's DNA fragments which have hybridized to the marker whether the subject's DNA displays a pattern of restriction fragment length polymorphisms consistently associated with a mutation at the Type II/III spinal muscular atrophy by determining whether the hybridized fragments resulting from the DNA isolated from the subject are the same in number and size as the hybridized fragments resulting from the DNA isolated from the normal member of the subject's family, or the hybridized fragments resulting from the DNA isolated from the member of the subject's family who suffers from Type II/III spinal muscular atrophy, thereby detecting> a mutation at the Type II/III spinal muscular atrophy locus.
  • This invention provides a polymorphic marker which maps to chromosomal region 5qll.2-13.3, in particular the marker designated D5S112 (ATCC ⁇ ccession No. ) .
  • This invention also provides a nucleic acid molecule isolated from an 11 centimorgan region 2 centimorgans proximal to the marker D5S6 and 4 centimorgans proximal o the m rker D5S78 in the 5qll.2-13.3 locus, in particular an isolated nucleic acid molecule which encodes a protein related to spinal muscular atrophy.
  • Figure 2 Four consanguineous, acute SMA families typed with DNA markers from 5qll.2-13.3 and the surrounding region.
  • families B, C, and D indicate the marriage of first cousins. All affected children have been diagnosed with acute SM ⁇ (SM ⁇ I ⁇ Werdnig-Hoffmann ⁇ severe ⁇ infantile) confirmed by electromyography (EMG) and muscle biopsy. Family ⁇ is a three-generation Irish family typed for two -affected children (V-5 and V-7). A third affected child died before blood samples could be obtained. Family B is North American. Individual III-2 is diagnosed as having chronic childhood-onset SMA. She was diagnosed at 16 months, never walked, and showed EMG and muscle biopsy characteristic of SMA. Individual IV-l was diagnosed with acute SMA, confirmed by EMG and muscle biopsy at six months and died at 24 months. Families C and D are from Israel.
  • FIG. 3 Family A is diagnosed with chronic SM ⁇ and family B wit., acute SM ⁇ . ⁇ ll affected individuals were confirmed by EMG and muscle biopsy analysis. The three affected children in family B are trizygotic triplets. Both families were typed with eight DN ⁇ markers and analysed with the other 14 chronic and acute families using the modified HOMOG program (13) .
  • the dotted line represents analysis of six acute families from Example 1. This analysis includes several additional data points and typing with one additional marJcer, D5S112.
  • the solid line represents analysis of the four consanguineous families in Figure 2.
  • the solid line represents the sum of the two separate analyses assuming genetic homogeneity between acute SMA families.
  • Marker JK53 (D5S112) was isolated from a flow-sorted library enriched for chromosome 5 (American Type Culture Collection) and mapped to 5qll.2-13.3 using hybrid cell lines (14). It detects a PvuII polymorphism with variable bands of I8kb
  • SMA Type I and SMA Types II/III acute and chronic childhood-onset SMA locus maps to an 11 centimorgan (11,000 kilobase) region located within the 5qll.2-13.3 region on chromosome 5q. Although acute and chronic SMA map to the same locus, they represent two different mutations at that locus.
  • This invention provides a method for determining whether a subject is likely to develop Type I spinal muscular atrophy by detecting a mutation at the Type I spinal muscular atrophy locus, which comprises a. isolating DNA from the subject, b. digesting the subject's isolated DN ⁇ with at least one restriction enzyme for a marker mapping to the locus 5qll.2-13.3 in close proximity to the Type I spinal muscular atrophy locus and which detects a restriction fragment length polymorphism with this enzyme which is consistently associated with a mutation at the Type I spinal muscular atrophy locus, c. electrophoresing the subject's resulting DN ⁇ fragments on a gel, d.
  • a detectable label to at least one marker mapping to the locus 5qll.2- 13.3 which has a restriction fragment length polymorphism for a restriction enzyme of step b consistently associated with a mutation at the Type I spinal muscular atrophy locus, e. contacting the subject's DN ⁇ fragments of step c with at least one detectably labelled marker of step d under conditions such that the detectably labelled marker hybridizes to the appropriate DN ⁇ fragments, f. visualizing the resulting detectably labeled DN ⁇ fragments, and g.
  • DNA is isolated from a sample of the subject's blood or tissue, -digested, and hybridized to a probe by methods well known in the art.
  • a procedure is as follows: The blood or homogenized tissue sample is centrifuged and the resulting cell pellet is lysed with a detergent. The lysate is exposed to enzymes which break down cell debris (proteins, lipids) and RN ⁇ , and the DN ⁇ is extracted with phenol and ethanol-precipitated. The DN ⁇ is then exposed to the restriction enzyme or enzymes of choice in the appropriate buffer (for example TBE) and the resulting digest is placed into wells on an agarose gel and electrophoresed to separate the bands.
  • the appropriate buffer for example TBE
  • the DN ⁇ on the gel is transferred to a nylon or nitrocellulose filter by contact, and is denatured with base (NaOH) to enable it to hybridize with the probe.
  • the detectable marker can be radioactively labeled, for example with ⁇ PtATP] which it has incorporated.
  • the marker hybridizes to any DN ⁇ bands which have the corresponding sequence, thus labeling the bands. Labeled bands are detected by placing a piece of X-ray film over the filter and allowing the labeled bands to develop and thus mark the film. The results are analyzed as described below.
  • the marker maps to an 11 centimorgan region 2 centimorgans proximal to the marker D5S6 and 4 centimorgans proximal to the marker D5S78 in the 5qll.2-13.3 locus.
  • This region has been determined by linkage analysis to be the locus of the gene associated with spinal muscular atrophy. (Examples of markers, and methods for isolating markers, are described in more detail below) .
  • marker genes which are genes that are located close to a gene of interest, can be isolated and used as probes.
  • the gene of interest may itself encode an abnormal phenotype, or may encode or predispose to an abnormal phenotype in one of its allelic forms, or the gene may encode an abnormal phenotype when present in mutant form.
  • the marker gene or genes occur polymorphically, i.e. are present in the population in multiple slightly different forms.
  • the marker gene is a useful genetic probe for the abnormality.
  • Each variant of the polymorphic marker gene has a different restriction fragment pattern when its isolated DN ⁇ is digested with a single restriction enzyme or panel of restriction enzymes.
  • polymorphisms in the areas of the marker gene which have nucleotide sequences which form cut sites for restriction enzymes For example, one form of the marker gene may have the sequence AATTC which forms the site for the enzyme EcoRI.
  • the form associated with the gene of interest may have in the same area a polymorphic sequence
  • the isolated DN ⁇ comprising the marker gene is digested with EcoRI by methods well known in the art, the first form of the marker will be cut at the site described and this cut will create a fragment of a length determined by the location of the next EcoRI site (assuming this is a single-enzyme digest) . The second form of the marker will not be cut at this site, therefore the fragment generated by the digest will be longer.
  • the DN ⁇ digest is run on an agarose or polyaeryla ide sizing gel and hybridized with the marker gene detectably labelled to act as a probe, the detectable band visualized on the gel will correspond to the length of the restriction fragments produced.
  • the mutant gene of interest is detected by detecting the restriction fragment pattern given by the polymorphism of the marker gene which is genetically linked to the mutant gene of interest.
  • a mutant gene is given only as an example. This method may be used to detect alleles and polymorphisms of a gene of interest, or the gene itself, not necessarily a mutant form of the gene. If the gene is
  • a carrier of the abnormality may not show an abnormal phenotype. Therefore a means to discover whether the gene is abnormal is important.
  • SMA carrier status cannot be determined using electromyography and muscle biopsy since SM ⁇ carriers are clinically normal.
  • Carrier status can only be detected using genetic analysis as described, using closely linked markers. Even if the gene is dominant thus always associated with an abnormal phenotype, a diagnostic test is useful to distinguish the specific abnormality from other abnormalities which have
  • This invention also provides a method for determining whether a subject is likely to develop Type II/III spinal muscular atrophy by detecting a mutation at the Type II/III spinal muscular atrophy locus, which comprises, a. isolating DNA from the subject, b. digesting the subject's isolated
  • the marker maps to an 11 centimorgan region 2 centimorgans proximal to the marker
  • the detectable marker can be radioactively labeled, for example with ⁇ PCATP].
  • a particularly useful test results when the subject is a fetus and the DNA is isolated from a niotic fluid.
  • This invention further provides a method for determining whether a subject is likely to develop Type I spinal muscular atrophy by detecting a mutation at the Type I spinal muscular atrophy locus, which comprises, a. isolating DN ⁇ from the subject, from a normal member of the subject's family, and from at least one member of the subject's family who suffers from Type I spinal muscular atrophy, b. digesting the isolated DN ⁇ s with at least one restriction enzyme for a marker mapping to the locus 5qll.2-l3.3 in close proximity to the Type I spinal muscular atrophy locus and which detects a restriction fragment length polymorphism consistently associated with a mutation at the Type I spinal muscular atrophy locus, c.
  • determining by the number and size of the DN ⁇ fragments which have hybridized to the marker which polymorphism is consistently associated with a normal genotype and which polymorphism is consistently associated with a mutation at the Type I spinal muscular atrophy locus in the genome of the subject's family by comparing the hybridized fragments resulting from the DN ⁇ isolated from the normal member of the subject's family and the hybridized fragments resulting from the DNA isolated from the member of the subject's family who suffers from Type I spinal muscular atrophy, and i.
  • determining by the number and size of the subject's DN ⁇ fragments which have hybridized to the marker whether the subject's DN ⁇ displays a pattern of restriction fragment length polymorphisms consistently associated with a mutation at the Type I spinal muscular atrophy by determining whether the hybridized fragments resulting from the DN ⁇ isolated from the subject are the same in number and size as the hybridized fragments resulting from the DN ⁇ isolated from the normal member of the subject's family, or the hybridized fragments resulting from the DNA isolated from the member of the subject's family who suffers from Type I spinal muscular atrophy, thereby detecting a mutation at the Type I spinal muscular atrophy- locus.
  • the marker maps to an 11 centimorgan region 2 centimorgans proximal to the marker D5S6 and 4 centimorgans proximal to the marker D5S78 in the 5qll.2-13.3 locus.
  • Polymorphisms in certain markers are associated with a propensity to develop SMA. ⁇ particular polymorphism is associated with normal or SMA DNA consistently within a single family, therefore it is necessary to test the DN ⁇ of members of the subject's family, as well as the subject's DN ⁇ , to determine which polymorphism of the marker is associated with SM ⁇ in the subject's family by comparing restriction patterns generated by normal and SM ⁇ -diagnosed family members.
  • the marker gene is a useful genetic probe for the abnormality if DN ⁇ can be collected from two affected family members per family. If a biopsy sample from the first affected family member demonstrates only the "long" fragment (see discussion supra of RLFP analysis) , then we can predict that inheritance of the long fragment in a second patient sample (from amniocentesis, for example) implies co-inheritance of the disease gene. Co- inheritance of a polymorphic variant that segregates with disease in a given family can be used to predict carrier status.
  • markers and their accompanying polymorphic restriction enzymes are used in this method. These markers are the marker designated D5S112 with the restriction enzyme Pvu II, the marker designated D5S6 with the restriction enzyme BamHI, and the marker designated D5S39 with the restriction enzyme Msp I. These markers can also be used simultaneously in a panel consisting of at least two of the markers designated D5S112 ( ⁇ TCC ⁇ ccession No. ) , D5S6
  • the detectable marker can be radioactively labeled, as described, for example with
  • This invention also provides a method for determining whether a subject is likely to develop Type II/III spinal muscular atrophy by detecting a mutation at the Type II/III spinal muscular atrophy locus, which comprises, a. isolating DN ⁇ from the subject, from a normal member of the subject's family, and from at least one member of the subject's family who suffers from Type II/III spinal muscular atrophy, b. digesting the isolated DN ⁇ s with at least one restriction enzyme for a marker mapping to the locus 5qll.2-13.3 in close proximity to the Type II/III spinal muscular atrophy locus and which detects a restriction fragment length polymorphism consistently associated with a mutation at the Type II/III spinal muscular atrophy locus, c.
  • a detectable label to at least one marker mapping to the locus 5qll.2-13.3 which has a restriction fragment length polymorphism for a restriction enzyme of step b consistently associated with a mutation at the Type II/III spinal muscular atrophy locus, e. contacting the DNA fragments of step c with at least one detectably labelled marker of step d under conditions such that the detectably labelled marker hybridizes to the appropriate DNA fragments, f. visualizing the resulting detectably labeled DNA fragments, g.
  • the marker maps to an
  • D5S39 BamHI, and the marker designated D5S39 with the restriction enzyme M ⁇ p I.
  • D5S6 detects a BamHI polymorphism with variable bands of 11 kb, 9.6 kb, and 7.6 kb.
  • D5S39 detects an Msp I polymorphism with variable bands of 8.7 kb and 5.8 kb.
  • D5S112 detects a Pvu II polymorphism with variable bands of 18 kb and 4.9 kb.
  • the detectable marker can be radioactively labeled, for example with 32 P[ ⁇ TP]_.
  • This invention provides a polymorphic marker which maps to chromosomal region 5qll.2-13.3, in particular the marker designated D5S112.
  • This marker has been deposited with the American Type Culture Collection under ⁇ TCC ⁇ ccession No. , pursuant to, and in satisfaction of, the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and were made with the American Type culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852.
  • Probes were screened for detection of restriction fragment length polymorphism by hybridization to panels of five unrelated individuals digested with each of ten different restriction enzymes. When RLFPs were detected, Mendelian inheritance was tested by hybridization to large reference pedigrees from the Centre D'Etude de Polymorphisms Humain (CEPH) collection. The pattern of allele segregation was then compared to other chromosome 5 markers and the data analyzed using the LINKAGE and LINKMAP programs (33, 20). Marker JK53 (D5S112) detects a PvuII polymorphism with variable bands of 18kb (44%) and 4.9kb (56%) and a constant 3. 5 kb band. Typed against CEPH reference pedigrees, it maps 0.5 cM telomeric of probe D5S6.
  • Markers progressively closer to the the spinal muscular atrophy gene are isolated by the method described above for JK53 and tested for linkage using DNA from families afffected by spinal muscular atrophy.
  • ⁇ minimal genetic region is defined by recombination between the set of markers and the gene. This region, containing the gene, is mapped by digesting with restriction enzymes and resolving the restiction digests using pulsed-field electrophoresis (an electrophoresis method well known in the art by which large fragments of DNA can be resolved by passing pulses of current instead of steady current through a gel) .
  • the fragments are cut out of the gel, and cloned into yeast artificial chromosomes and co ⁇ mids (both able to accept large DNA fragments) for manipulation, by methods well known in the art.
  • the clones are ordered based on restriction digests (matching neighboring sites on different clones) to construct a map or the region.
  • the markers are hybridized with the clones to locate them on the restriction map.
  • ⁇ set of clones as described is prepared from normal DN ⁇ and from DN ⁇ of an individual affected with spinal muscular atrophy. The maps can be compared for differences in restriction sites to determine more markers.
  • the clones are sequenced, and differences in sequence between normal and affected DN ⁇ are determined.
  • an allele-specific oligonucleotide 30 to 40 nucleotides long is prepared for each mutation isolated, having the same sequence as the mutation. This oligonucleotide is used to probe fresh samples of spinal muscular atrophy DN ⁇ . Its hybridization indicates that its sequence represents the mutant sequence. Thereby the gene is localized and is isolated from the clones already prepared, mapped, and sequenced.
  • Age of onset in chronic families was 2-10 years, and symptoms ranged from death in adolescence to only minor impairment of function, with affected individuals meeting criteria for either the intermediate or mild forms of childhood-onset SMA (types II and III) (4) .
  • Age of onset in acute cases ranged from birth to 6 months, and age at death ranged from 8 months to 2 years.
  • One child has survived to 5 years of age with the aid of a respirator. Because of the aggressive nature of acute SM ⁇ , it is difficult to locate families with many living affected children. ⁇ ll 13 families meet diagnostic criteria for SM ⁇ .
  • DN ⁇ fragment length polymorphisms 5 ⁇ g of each DN ⁇ was digested to completion with the indicated restriction enzyme as outlined by the supplier (Boehringer-Mannheim, New England Biolabs) .
  • the resulting DN ⁇ fragments were resolved according to size by horizontal gel electrophoresis in TBE buffer, and, after staining with ethidium bromide for direct visual inspection, the DN ⁇ fragments were denatured in situ in the gel with l N NaOH.
  • the denatured DN ⁇ fragments were transferred to a nylon filter support (Zetapor, ⁇ MF/Cuno, Meriden, re ⁇ connecticut) by Southern blotting, and fixed to the filter by baking.
  • DNA probes were labeled with [ 32 P]-ATP (Amersham) by random oligonucleotide priming (31) , and then hybridized to the filter for 48 hours at 65° C in 6x SSC, lx Denhardf's, 0.3% SDS, and 100 ⁇ g/ml of salmon testis DNA.
  • the filters were washed in 0.5x SSC at 65 ⁇ C and exposed to X-ray film (Kodak XAR-5) with a Dupont Cronex intensifying screen at -80° C for 48 hours. Alleles present in each individual were deduced from the sizes of the bands on the developed film.
  • the peak multipoint lod score for chronic SM ⁇ is 9.03, and the peak lod score for acute SM ⁇ is 2.02.
  • Pairwise lod scores for chronic and acute SM ⁇ families versus four markers located in the middle of the linkage region are shown in Table 1.
  • the maximum two-point lod score for chronic families is 8.43 at a recombination fraction of 2% with marker D5S6 ( ⁇ TCC Catalog Nos. 57686-7), and 1.71 for acute families at a recombination fraction of 2% with marker D5S78 (ATCC Catalog Nos. 59308-9) .
  • the confidence interval for the location of the gene for chronic SM ⁇ is 11 centimorgans (cM) wide and spans a region of 2 cM proximal of locus D5S6 to a point 4cM proximal of locus D5S78 (note arrows in Fig. 1) .
  • the maximum lod score of 2.02 indicates a gene responsible for those disease maps to the same general area.
  • the best estimate for the location of the acute SM ⁇ locus is 15 cM distal to the estimated position of the locus for chronic SM ⁇ .
  • the chronic forms of childhood- onset SM ⁇ therefore, are likely to occur as the result of allelic heterogeneity, similar to the case for Duchenne and Becker-type dystrophies (15).
  • Example 2 demonstrates that adult-onset and dominantly inherited cases of SM ⁇ similarly map to chromosomes 5q.
  • Chronic SMA refers to cases which meet diagnosic criteria for intermediate (SMA type II) and mild (Kugelberg-Welander, SMA type III) forms.
  • Acute SMA refers to cases which meet diagnosic criteria for SMA type I (Werdnig-Hoffmann, infantile SMA, severe SMA) .
  • Figure 2 shows the DN ⁇ marker typing for four consanguineous, acute SM ⁇ families. ⁇ s a result of inbreeding, five typed individuals in family A are most likely obligate carriers of a common disease locus, and thus provide the opportunity to score multiple recombination events with the disease. The parents of the affected children in families B, C, and D are related as first cousins. Family B contains a mother diagnosed with chronic SM ⁇ and her child who is diagnosed with acute SM ⁇ . nux ⁇ ipoin'c lod scores for the families in Figure 2 were calculated and the evidence for heterogeneity within the acute and chronic groups evaluated by allowing for an admixture of linked and unlinked families in the usual manner (Table 2) .
  • the map location of the disease locus is constrained to be at the same point, x, for the two groups; at each x value, the log(L) is maximized over ⁇ in each group and added for the two groups; the maximum log(L) over the points x occured at the same value as in the families with acute SM ⁇ .
  • the antilog of the difference (11.41-11.21 - 0.20) is equal to 1 .6 and represents the likelihood ratio for heterogeneity between the two family groups. Initially, individual III-2 in family B was classified as unaffected to allow for heterogeneity between the two groups. The likelihood ratios in favor of heterogeneity are
  • D5S76 and D5S78 The affected child in family D seems heterozygous throughout this region, but is not identified as "unlinked" by the HOMOG program.
  • Family B is of interest because the mother (III-2) is diagnosed with chronic SM ⁇ , and the child (IV-1) with acute SM ⁇ .
  • a disease allele from generation I is transmitted to both carrier parents in generation III, then inherited in double copy by individual IV-1.
  • the affected child is homozygous for markers D5S39 and D5S78.
  • the affected mother presumably has one copy of the disease allele in common with the affected child, and one heterologous copy inherited from her father.
  • this family is consistent with all ⁇ lic variation among chronic and acute cases of SM ⁇ .
  • the affected mother could have transmitted a sporadic dominant mutation for SMA, though this is unusual in cases of childhood-onset SMA.
  • the nine acute and seven chronic SM ⁇ families in this and Example 1 provide strong evidence in favor of genetic homogeneity between the two disorders.
  • One chronic and one acute family appear to be unlinked to 5q markers, although family ⁇ can provide data in support of linkage when more informative markers are discovered.
  • the evidence for genetic homogeneity combined with linkage data indicate that the acute form of SM ⁇ maps to 5q.
  • SMA can be confused with clinically overlapping neurological disorders (1-4, 23), and the subdivision between acute and chronic SM ⁇ allows some overlap between the groups (29) . Therefore a genetic screen for these conditions is diagnostically very useful.

Landscapes

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

Abstract

Procédés de détection d'atrophie musculaire spinale de type I et de types II/III consistant à utiliser des marqueurs présentant des polymorphismes de longueur de fragment de restriction, marqueur utilisé dans la détection de l'atrophie musculaire spinale de type I et des types II/III, et molécules d'acide nucléique isolées associées à l'atrophie musculaire spinale.
PCT/US1991/004605 1990-06-27 1991-06-27 Procedes de detection d'atrophie musculaire spinale de type i et de types ii/iii et marqueur a cet effet WO1992000386A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US54440590A 1990-06-27 1990-06-27
US544,405 1990-06-27

Publications (1)

Publication Number Publication Date
WO1992000386A1 true WO1992000386A1 (fr) 1992-01-09

Family

ID=24172039

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/004605 WO1992000386A1 (fr) 1990-06-27 1991-06-27 Procedes de detection d'atrophie musculaire spinale de type i et de types ii/iii et marqueur a cet effet

Country Status (2)

Country Link
AU (1) AU8196791A (fr)
WO (1) WO1992000386A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2720757A1 (fr) * 1994-06-03 1995-12-08 Inst Nat Sante Rech Med Procédé et sondes pour la détection de marqueurs liés au locus des amyotrophies spinales infantiles.
EP0708178A1 (fr) * 1994-10-19 1996-04-24 Institut National De La Sante Et De La Recherche Medicale (Inserm) Gène de la survie des neurones moteurs: gène de l'atrophie musculaire spinale
WO1996012016A1 (fr) * 1994-10-18 1996-04-25 The University Of Ottawa Proteine inhibitrice de l'apoptose neuronale, sequence et mutations geniques induisant l'atrophie musculaire spinale
EP0711833A3 (fr) * 1994-10-19 1997-03-05 Inst Nat Sante Rech Med Gène de survie des neurones moteurs: gène de l'atrophie musculaire spinale
US9328346B2 (en) 2010-11-12 2016-05-03 The General Hospital Corporation Polycomb-associated non-coding RNAs
US9920317B2 (en) 2010-11-12 2018-03-20 The General Hospital Corporation Polycomb-associated non-coding RNAs
US10059941B2 (en) 2012-05-16 2018-08-28 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
US10174328B2 (en) 2013-10-04 2019-01-08 Translate Bio Ma, Inc. Compositions and methods for treating amyotrophic lateral sclerosis
US10655128B2 (en) 2012-05-16 2020-05-19 Translate Bio Ma, Inc. Compositions and methods for modulating MECP2 expression
US10837014B2 (en) 2012-05-16 2020-11-17 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
US10858650B2 (en) 2014-10-30 2020-12-08 The General Hospital Corporation Methods for modulating ATRX-dependent gene repression
US10900036B2 (en) 2015-03-17 2021-01-26 The General Hospital Corporation RNA interactome of polycomb repressive complex 1 (PRC1)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0317239A2 (fr) * 1987-11-13 1989-05-24 Native Plants Incorporated Procédé et dispositif pour la détection des polymorphismes de restriction des longueurs de fragments

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0317239A2 (fr) * 1987-11-13 1989-05-24 Native Plants Incorporated Procédé et dispositif pour la détection des polymorphismes de restriction des longueurs de fragments

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Biosis, Abstract No. 84,076,716, issued 1987, BOLHUIS et al., "Differential Diagnosis of Genetic Disease by DNA Restriction Fragment Length Polymorphisms"; & CLIN. CHIM. ACTA, 165 (2-3). *
Biosis, Abstract No. 88,026,334, issued 1989, DEFESCHE et al., "DNA Restriction Fragment Lenght Polymorphisms in Differential Diagnosis of Genetic Disease Application in Neuromuscular Diseases"; & HUM. GENET. 82 (1), p. 55-58. *
MEDLINE, Abstract No. 88,181,067, issued February 1988, WOOD et al., "A grandpaternally derived de novo deletion within Xp21 initially presenting in carrier females diagnosed as Kugelberg-Welander Syndrome", AM. J. MED. GENET., 29(2), p. 419-23. *
World Patent Index, Accession No. 89-152,719/21, issued 24 May 1989, HELENTJARI et al., "Rapid Restriction Fragment Polymorphism Analysis, esp. in Plant Study Using a Primer Labelled with a Fluorescing Molecule and an Elongating Enzyme"; & EP,A,317 239. *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2720757A1 (fr) * 1994-06-03 1995-12-08 Inst Nat Sante Rech Med Procédé et sondes pour la détection de marqueurs liés au locus des amyotrophies spinales infantiles.
WO1995033852A1 (fr) * 1994-06-03 1995-12-14 Institut National De La Sante Et De La Recherche Medicale Procede et sondes pour la detection de marqueurs lies au locus des amyotrophies spinales infantiles
US6040142A (en) * 1994-06-03 2000-03-21 Institut National De La Santa Et De La Recherche Medicale Method and probes for detecting markers linked to the infantile spinal muscular atrophy locus
WO1996012016A1 (fr) * 1994-10-18 1996-04-25 The University Of Ottawa Proteine inhibitrice de l'apoptose neuronale, sequence et mutations geniques induisant l'atrophie musculaire spinale
US6020127A (en) * 1994-10-18 2000-02-01 The University Of Ottawa Neuronal apoptosis inhibitor protein, gene sequence and mutations causative of spinal muscular atrophy
US6429011B1 (en) 1994-10-18 2002-08-06 University Of Ottawa Neuronal apoptosis inhibitor protein gene sequence and mutations causative of spinal muscular atrophy
EP0708178A1 (fr) * 1994-10-19 1996-04-24 Institut National De La Sante Et De La Recherche Medicale (Inserm) Gène de la survie des neurones moteurs: gène de l'atrophie musculaire spinale
EP0711833A3 (fr) * 1994-10-19 1997-03-05 Inst Nat Sante Rech Med Gène de survie des neurones moteurs: gène de l'atrophie musculaire spinale
US9856479B2 (en) 2010-11-12 2018-01-02 The General Hospital Corporation Polycomb-associated non-coding RNAs
US10358644B2 (en) 2010-11-12 2019-07-23 The General Hospital Corporation Polycomb-associated non-coding RNAs
US9328346B2 (en) 2010-11-12 2016-05-03 The General Hospital Corporation Polycomb-associated non-coding RNAs
US9920317B2 (en) 2010-11-12 2018-03-20 The General Hospital Corporation Polycomb-associated non-coding RNAs
US10053694B2 (en) 2010-11-12 2018-08-21 The General Hospital Corporation Polycomb-associated non-coding RNAS
US11066673B2 (en) 2010-11-12 2021-07-20 The General Hospital Corporation Polycomb-associated non-coding RNAs
US10119144B2 (en) 2010-11-12 2018-11-06 The General Hospital Corporation Polycomb-associated non-coding RNAs
US9816094B2 (en) 2010-11-12 2017-11-14 The General Hospital Corporation Polycomb-associated non-coding RNAs
US10837014B2 (en) 2012-05-16 2020-11-17 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
US10655128B2 (en) 2012-05-16 2020-05-19 Translate Bio Ma, Inc. Compositions and methods for modulating MECP2 expression
US10059941B2 (en) 2012-05-16 2018-08-28 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
US11788089B2 (en) 2012-05-16 2023-10-17 The General Hospital Corporation Compositions and methods for modulating MECP2 expression
US10174328B2 (en) 2013-10-04 2019-01-08 Translate Bio Ma, Inc. Compositions and methods for treating amyotrophic lateral sclerosis
US10858650B2 (en) 2014-10-30 2020-12-08 The General Hospital Corporation Methods for modulating ATRX-dependent gene repression
US10900036B2 (en) 2015-03-17 2021-01-26 The General Hospital Corporation RNA interactome of polycomb repressive complex 1 (PRC1)

Also Published As

Publication number Publication date
AU8196791A (en) 1992-01-23

Similar Documents

Publication Publication Date Title
Murray et al. Linkage of an autosomal dominant clefting syndrome (Van der Woude) to loci on chromosome Iq
Los et al. The importance of genetic influences in asthma
Antonarakis et al. Genetic diseases: diagnosis by restriction endonuclease analysis
US5576178A (en) Method of detecting genetic deletions and mutations associated with DiGeorge syndrome, Velocardiofacial syndrome, charge association, conotruncal cardiac defect, and cleft palate and probes useful therefor
US4965190A (en) Methods for the identification of mutations in the human phenylalanine hydroxylase gene using DNA probes
Fujita et al. Analysis of the RPGRGene in 11 Pedigrees with the Retinitis Pigmentosa Type 3 Genotype: Paucity of Mutations in the Coding Region but Splice Defects in Two Families
WO1992000386A1 (fr) Procedes de detection d'atrophie musculaire spinale de type i et de types ii/iii et marqueur a cet effet
Feder et al. DNA restriction fragment analysis of the proopiomelanocortin gene in schizophrenia and bipolar disorders
Kenney et al. Characterization of the p67phox gene: genomic organization and restriction fragment length polymorphism analysis for prenatal diagnosis in chronic granulomatous disease
Whaley et al. Mapping of D4S98/S114/S113 confines the Huntington's defect to a reduced physical region at the telomere of chromosome 4
Sheth et al. Linkage analysis in spinal muscular atrophy, by six closely flanking markers on chromosome 5
US6303294B1 (en) Methods of detecting genetic deletions and mutations associated with Digeorge syndrome, Velocardiofacial syndrome, CHARGE association, conotruncal cardiac defect, and cleft palate and probes useful therefore
CA2136859C (fr) Utilisation de marqueurs genetiques pour le diagnostic de la dysautonomie familiale
Pritchard et al. Recombination of 4p16 DNA markers in an unusual family with Huntington disease
JPH10500860A (ja) 小児脊髄性筋萎縮症の遺伝子座に連結したマーカーの検出方法およびプローブ
CA2306194C (fr) Chromosome 9 et marqueurs genetiques de la degenerescence progressive des cones et des batonnets, et analyses correspondantes
Farrall et al. Cystic fibrosis carrier detection using a linked gene probe.
Hart et al. The screening of Duchenne muscular dystrophy patients for submicroscopic deletions.
Zschocke et al. Large heterozygous deletion masquerading as homozygous missense mutation: a pitfall in diagnostic mutation analysis
Onuchic et al. Sequence analysis of the human hTg737 gene and its polymorphic sites in patients with autosomal recessive polycystic kidney disease
Hill et al. The Mendelian inheritance of a human X chromosome-specific DNA sequence polymorphism and its use in linkage studies of genetic disease
Roses Molecular genetics of myotonic and Duchenne muscular dystrophies
Morgan et al. The high frequency of the—6G→ A factor IX promoter mutation is the result both of a founder effect and recurrent mutation at a CpG dinucleotide
Klinger Genetics of cystic fibrosis
WO1993017104A1 (fr) Sequence adn du gene de dystrophie myotonique et ses utilisations

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU 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 IT LU NL SE

NENP Non-entry into the national phase

Ref country code: CA