WO1993011149A1 - Methods of detecting a genetic predisposition for osteoporosis - Google Patents

Methods of detecting a genetic predisposition for osteoporosis Download PDF

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Publication number
WO1993011149A1
WO1993011149A1 PCT/US1992/010355 US9210355W WO9311149A1 WO 1993011149 A1 WO1993011149 A1 WO 1993011149A1 US 9210355 W US9210355 W US 9210355W WO 9311149 A1 WO9311149 A1 WO 9311149A1
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dna
type
sequence
osteoporosis
kit
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PCT/US1992/010355
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French (fr)
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Darwin J. Prockop
Loretta D. Spotila
Constantinos D. Constantinou
Larisa Sereda
Andrea Westerhausen
Michael Pack
Alain Colige
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Thomas Jefferson University
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    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • 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

  • the present invention relates to the field of methods for detecting genetic diseases and more particularly to the field of methods for detecting genetic diseases linke to anomalies of genes for collagens and other structural proteins found in bone.
  • Post-menopausal osteoporosis is an important cause of serious disability.
  • the disease is usually defined as a condition in which there is marked decrease in bone mass (osteopenia) associated with one or more fractures from relatively minor trauma.
  • osteoopenia bone mass associated with one or more fractures from relatively minor trauma.
  • Two forms of the disease have been described: Type I or post-menopausal osteoporosis that primarily affects women within 10 to 15 years after menopause, and type II or age-related osteoporosis that occurs in both men and women over the age of 70.
  • Type I osteoporosis is apparently triggered by the transient acceleration of bone loss that occurs with the fall in estrogen levels of women at menopause. It is believed that the disease is familial.
  • Bone loss that characterizes osteoporosis cannot be effectively reversed by any known therapies.
  • the rate of bone loss can be decreased by administration of estrogens and several other agents. It is generally not advisable to administer estrogens and related agents in large doses that are needed for their effectiveness to all individuals because the therapies have side effects such as feminizing effects in men and increasing the chances of cervical and breast cancer in women.
  • women predisposed to type I osteoporosis can be identified early, it could be justifiable to accept the risk of administering estrogens or related agents and to monitor carefully for the early development of cancers so as to avoid the devastating effects of the osteoporosis itself.
  • Bone is a complex structure whose strength and resistance to fractures depends on a number of factors.
  • a major source of the strength of bone are fibrils of type I collagen that form a scaffold on which the mineral of bone is deposited.
  • the proper deposition of mineral on the collagen fibrils of bone probably depends other structural proteins such as osteopontin that bind to the collagen fibrils and participate in the mineralization process.
  • Type I collagen is a member of a family of fibrillar collagens and accounts for 80 to 90% of the protein found in bone. It is also found in large amounts in tissues such as skin, ligaments and tendons.
  • Type I collagen is first synthesized as a precursor known as type I procollagen that is formed from two identical pro ⁇ l(I) chains and one slightly different pro ⁇ 2(I) chain. Each chain contains three separate domains.
  • the N-propeptide domain at one end of a pro ⁇ chain contains a globular subdomain, a short triple-helical subdomain and another short subdomain that forms part of the cleavage site to the removal of the N-propeptide.
  • the C-propeptide domain at the other end of a pro ⁇ chain is entirely globular.
  • each pro ⁇ chain contains about 1,000 amino aci residues, and with the exception of a short seguence at the end of the chains, every third amino acid is glycine.
  • the molecular formula of an ⁇ chain can be represented as (-Gly-X-Y-) , where X- and Y- positions denote amino acids other than glycine.
  • X- and Y- positions denote amino acids other than glycine.
  • the presence of glycine, th smallest amino acid, in every third position is critical, since the amino acid in this position fits into a restricted space in which the three chains come together in the center of the triple helix.
  • the X- and Y- positions are frequently occupied by proline and 4-hydroxyproline, respectively.
  • the biosynthesis of the procollagen molecule involves a large number of post-translational modifications that require at least eight specific enzymes and several non-specific enzymes. In total, over 100 amino acids in each a chain are modified.
  • the pro ⁇ l(I) chain of type I procollagen is synthesized on one gene (COLlAl) found in chromosome 17.
  • the pro ⁇ 2(I) chain of type I procollagen is synthesized on the second gene (C0L2A1) found in chromosome 7.
  • osteogenesis imperfecta a genetic disease of children characterized by brittleness of bone. Many but not all children with osteogenesis imperfect also have blueness of the sclerae of the eyes, poor dentitio and thin skin because of a decrease in the amount of type I collagen or because of the formation of abnormal type I collagen fibrils.
  • the brittleness of bone seen in osteogenesis imperfecta is usually apparent early in childhood because the patients develop many fractures for minor trauma. Many of the patients with mild forms of the disease become fracture-free after the growth spurt of puberty but then develop a marked tendency to fracture later in life.
  • Mutations in the gene for type II procollagen have been shown to cause genetic disorders (chondrodysplasias) of cartilage, a tissue that is rich in this protein.
  • Mutations in the gene for type III procollagen have been shown to caus the type IV variant of Ehlers-Danlos syndrome that is characterized by thinness and other abnormalities of skin together with sudden rupture of the aorta and other hollow organs that are rich in type III collagen.
  • Mutations in the gene for type III procollagen have recently been shown to also cause familial vascular aneurysms in families without any evidence of a genetic disease such as the Ehlers-Danlos syndrome or the Marfan syndrome.
  • Figure 1 is a schematic diagram of the 3'-half of the human pro ⁇ l(I) gene.
  • Figures 2A and 2B show the nucleotide sequences of the human COLlAl gene extending from intron 25 to exon 52.
  • Capital letters indicate terminal codons of exon sequences o five ambiguous bases in introns.
  • the alternative bases in some cloned PCR products were G/T at position +185 in intron 46; A/C at position +291 in intron 46; T/C at position +112 in intron 49; A/G at position +126 in intron 49; and C/T at position +179 in intron 50.
  • the present invention provides methods of screening persons to determine whether those persons have an increased likelihood of developing osteoporosis. Persons not otherwis known to have a connective tissue disease are screened to detect mutations in the two genes for type I procollagen tha indicate increased likelihood of developing osteoporosis.
  • a tissue sample is provided an the nucleotide base sequence of at least a portion of the DN for the pro ⁇ l(I) and the pro ⁇ 2(I) chain derived from the tissue sample is determined.
  • the DNA for the chains can be genomic DNA or cDNA prepared from mRNA.
  • nucleotide base sequence of the two genes for type I procollagen (COLlAl and C0L1A2) are then compared to the nucleotide base sequence of standard DNA sequence for the same two genes to determine differences in nucleotide bases at corresponding regions of the DNA.
  • a difference in the base sequence of the DNA from test sample as compared with a standard sequence indicates a increased likelihood of the mammal suffering from osteoporosis.
  • the methods of the invention make it possible to detect mutations in the two genes for type I procollagen in persons not otherwise known to have a connective tissue disease.
  • the methods of the invention also make it possible to diagnose collagen diseases that are reflected by mutations in the type I procollagen genes such as osteogenesis imperfecta, in persons who do not exhibit symptoms of the disease and who have not been diagnosed by physical symptoms of the disease (because symptoms are variable, also indicative of other diseases, or too mild for clinical or physical diagnosis to be made) . While some forms of osteogenesis imperfecta can be diagnosed by the presence of physical symptoms alone, the appearance of the disease in the general population may be more variable, with its presence in some persons and families remaining undetected because of the mildness or variability of the physical symptoms. In such cases, the methods of the invention may be particularly useful.
  • the methods of the invention are also useful for detecting genetic familial predisposition to osteoporosis.
  • the location of the mutation in either the gene for the pro ⁇ l(I) chain or the gene for the pro ⁇ 2(I) chain of a first family member known or suspected of having osteoporosis is determined.
  • the nucleotide sequence of at least the mutated region is then compared to the corresponding region in the same gene of a second family member, whereby the presence in the second family member of the mutated region indicates an increased likelihood of osteoporosis in the second family member.
  • one part of the present invention is to detect mutations in genes for other structural proteins of bone that cause osteoporosis.
  • the methods of the invention have two stages.
  • a person who has developed osteoporosis or is suspected of having or developing osteoporosis is tested to determine if he or she has a mutation in the DNA sequence of the pro ⁇ l(I) or pro ⁇ 2(I) gene (COLlAl or COL1A2) .
  • the comparison is done by comparing the corresponding regions of the DNA sequence from the person tested with a standard DNA sequence of the same genes.
  • the DNA sequence of the two genes from the person tested can be genomic DNA or cDNA prepared from RNA derived from a tissue sample taken from t person.
  • a standard DNA sequence for the genes can be obtained by reference of known sequences or to those set forth herein in Figures 1 and 2 and listed in the sequence listing. A difference in the base sequence of the DNA from the person tested as compared with the standard sequence indicates an increased likelihood of the mammal suffering from osteoporosis.
  • For the first family member tested all or a substantial portion of DNA coding for the two genes is sequenced and compared to a standard sequence. Sequencing o the first family member's DNA may be done by conventional DN sequencing techniques such as in Example 1.
  • the mutation in the gene is expected to appear in th same position in the pro ⁇ l(I) or pro ⁇ 2(I) gene.
  • Family A the genetic mutation may be at position 30; and for Family B the genetic mutation may be at position 550.
  • Testing the family members can be done by comparing corresponding regions of the family member's genes and the mutation to determine if the mutation is present in the family members in the second stage of the methods of the invention.
  • family members means persons genetically related to one another in any degree, such as parent-child, siblings, cousins, etc.
  • DNA is extracted from a test sample of cells of the family members to be tested by conventional techniques that involve lysis of the cells with sodium dodecyl sulfate (SDS) and digestion of proteins with proteinase K followed by extraction with phenol and chloroform, and ethanol precipitation as described in Maniatis et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982) , pp. 280-281.
  • a sample of cells can be taken from many types of tissues, such as a piece of skin, a sample of blood, or by scraping of the interior of the mouth.
  • mRNA can be extracted from the test sample and cDNA synthesized with reverse transcriptase, and the resulting cDNA used for analysis.
  • the DNA derived from the cells of the test sample is then analyzed to determine whether or not the pro ⁇ l(I) or pro ⁇ 2(I) gene for type I procollagen contains a mutation.
  • I a mutation is found in either of the two genes, a rapid test can be devised to test other members of the patients' families to determine whether or not they have the same mutation.
  • Family members who have the mutation can then be followed by techniques such as x-ray or photon absorption fo development of the osteopenia that is an early sign of osteoporosis and treated with estrogen or related agents so as to decrease the normal rate of bone loss with age.
  • the treatment can be initiated before there is evidence of a marked decrease in bone mass.
  • sequences for introns 25 to 51 presented in Figure 2 are part of the present invention. These sequences for introns 25 to 51 are important for developing oligonucleotide primers to amplify and sequence genomic DNA from patients suspected of having mutations in the gene for pro ⁇ l(I) chain of type I procollagen that cause osteoporosis.
  • Oligonucleotide primers for amplifying and sequencing the cDNA for the human pro ⁇ l(I) chain of type I procollagen has been published by Labhard and Hollister (1990) Matrix, 10:124-130. Primers for amplifying the cDNA for the human pro ⁇ l(I) chain are presented in Table 1 and are part of the present invention.
  • the same experimental techniques for detecting mutations in the two genes for type I procollagen can readily be applied to detecting mutations in genes coding for other structural proteins found in bone.
  • the nucleotide sequences of the osteopontin gene can be used to design oligonucleotide primers to amplify genomic DNA or cDNA for the gene with the polymerase chain reaction.
  • the products obtained from the polymerase chain reaction can then be used to define the base sequences of the gene itself or the cDNA. Therefore, mutations in the genes for these other structural proteins that cause osteoporosis can be detected in the same manner as mutations in the two genes for type I procollagen.
  • RNA including messenger RNA
  • RNA was isolated from cultured skin fibroblasts b lysis of the cells with the detergent Sarcosyl in the presence of guanidinium isothiocyanate and pelleting the RNA through cesium chloride according to the method of Maniatis et al., supra, p. 196.
  • cDNA was synthesized from the poly(A)+RNA, using a kit purchased from BRL (Bethesda Research Laboratories, Bethesda, MD) or Pharmacia (Pharmacia-LKB, Piscataway, NJ) or reverse transcriptase and a primer specific for the pro ⁇ l(I) chain such as those provided in Table 1.
  • Double-stranded cDNA was synthesized according to the method of Gubler and Hoffman (1983) Gene, 25:263-269 as amended by the manufacturers of the cDNA kits.
  • Single-stranded cDNA was synthesized using reverse transcriptase followed by alkaline hydrolysis of the RNA according to Maniatis supra, pp. 214-216.
  • the double- or single-stranded cDNA was amplified using a polymerase chain reaction kit (GeneA p DNA Amplification Reagent Kit, Perkin Elmer Cetus, Norwalk, CT) according to instructions provided by the supplier.
  • Primers complementary to different portion of the pro ⁇ l(I) or pro ⁇ 2(I) gene were used in the polymerase chain reaction.
  • the primers used are listed in Table 1.
  • Several different combinations of primers can be used to generate DNA containing mRNAs for type I procollagen.
  • PCR was carried out with asymmetric ratios of two primers so as to generate an excess of single-stranded DNA.
  • the single-stranded DNA was then used as template to determine the nucleotide sequence with the dideoxynucleotide chain termination method using internal primers and a sequencing kit (Sequenase; United States Biochemical Corp. , Cleveland, OH) .
  • the cDNA was used as template for polymerase chain reaction with primers in Table 1 at 94°C for 1.5 minutes, 56°C or 58°C for 1 minute and 74"C for 1.5 minutes.
  • the picomolar ratio of the forward primer to the reverse primer was 20:4 and 30 cycles of amplification were performed.
  • 1/100 of the first polymerase chain reaction product was used as template for 20 cycles in which the picomolar ratio of the forward or reverse primers were 50:1.
  • the final product was purified, and the volume reduced using Ultrafree MC filtration units (Millipore No. UFC3TTK00) .
  • the purified DNA was then used directly for the dideoxynucleotide chain reaction, as described by Sanger et al. (1977) Proc. Nat. Acad. Sci. USA, 74:5463-5467, using modified T7 DNA polymerase (United States Biochemical Corp., Cleveland, OH) . Both conventional and radioactive sequencin using S-dATP autoradiography and sequencing using fluorescently labeled primers and an AB1 370A automated sequencer (Applied Biosystems International, San Francisco, CA) were used to determine the sequences.
  • the sequences from the first family member are compared with the standard DNA sequence of th gene such as the sequences in Figure 2. Changes in the bas sequence of tested family members indicated an increased likelihood of that person developing osteoporosis.
  • Example 2 Isolation and Characterization of Nucleotide Sequences of Introns 25 to 51 of the Pro ⁇ l(I) Gene
  • deletion library was prepared with exonuclease III and a commercial kit (Erase-a-base, Stratagene) .
  • Double-stranded DNA from deletion library was used for sequencing with the dideoxynucleotide procedure (Sanger et al. (1977) PNAS 74:5463-5467) and T7 polymerase (Sequenase; U.S. Biochemicals) .
  • Skin fibroblasts were examined from a 52-year old woman with post-menopausal osteoporosis or type I osteoporosis. The woman was Caucasian and was evaluated at the Mayo Clinic after she had developed acute mid-thoracic pain following a severe jolt while driving a truck. X-ray examination of the spine showed an anterior compression fracture of the ninth thoracic vertebra and generalized demineralization of the spinal column consistent with osteoporosis.
  • Bone densitometry of the lumbar spine assayed by dual-energy X-ray absorptiometry was 0.75 g/cm , a value that was in the lowest second percentile for the same age and sex (mean normal value 1.13 g/cm ) .
  • the sclerae had a slightly bluish cast, the skin was not abnormally thin, and there is no hyperextensibility of joints. Routine laboratory tests were normal, including serum protein electrophoresis. The patient had a normal menopause seven years earlier. There was no history of any disease or use of drug known to be associated with osteoporosis.
  • Type I procollagen synthesized by the patient's dermal fibroblasts was examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. There was delaye migration in both the ⁇ l(I) and ⁇ 2(I) chains derived from th secreted type I procollagen. The difference was more apparent when the vertebrate collagenase fragments were examined.
  • RNA was prepared from the patient's fibroblasts and used to synthesize first-strand cDNA.
  • the single-stranded cDNA was then used as a template for nine polymerase chain reactions using primers that amplified all 3,052 base pairs of codingsequence for the triple-helical domains of the pro ⁇ l( and pro ⁇ 2(I) chain.
  • Each polymerase chain reaction product was analyzed by a procedure in which heterozygous single-bas mutations can be detected by denaturing and renaturing the products to form heteroduplexes and then treating the heteroduplexes with a water-soluble carbodiimide (Ganguly an Prockop (1990) Nucleic Acids Research, 18:3933-3939).
  • Analysis of the nine polymerase chain reaction products by primer extension suggested that only one contained a sequenc variation.
  • the region of interest spanned nucleotides 1,951 to 2,813 (amino acids 516 to 803 of the triple-helical domain) of the ⁇ 2(I) coding sequence.
  • the results suggested that a sequence variation was present in the region encoding amino acid residues 660 to 667 of the ⁇ 2(I) chain.
  • the patient's genomic DNA was used as a template for the polymerase chain reaction.
  • the polymerase chain reaction products were then hybridized with allele-specific oligonucleotides for the normal coding sequence, or for the normal coding sequence with a single-base substitution that converted the codon for glycine-661 of the ⁇ 2(I) chain to a codon for serine.
  • Both oligonucleotides hybridized with polymerase chain reaction products prepared from the patient's genomic DNA.
  • the oligonucleotide with a serine codon did not hybridize with polymerase chain reaction products from 50 control samples from genomic DNA.
  • other family members can be teste to determine if they also have mutations in the same gene that predisposes them to diseases such as post- menopausal osteoporosis. Testing of other family members can readily b done by probing the family members' DNA with a nucleotide probe having a base sequence complementary to the mutation i the first family member.
  • the DNA from other family members can be genomic DNA amplified with the polymerase chain reaction or cDNA synthesized by conventional techniques usin reverse transcriptase and mRNA templates.
  • a preferred test format is a nucleic acid hybridization assay such as dot or slot blot assays or Southern transfer of DNA fragments after separation on agarose gel.
  • Other methods such as restriction endonuclease digestion of amplified DNA followed by agarose gel electrophoresis and visualization of the DNA by ethidium bromide, if the mutation has created or destroyed a restriction endonuclease recognition site.
  • a DNA probe having a sequence which includes the mutation in the first family member's pro ⁇ l(I) or pro ⁇ 2(I) genes is synthesized using standard techniques.
  • the probe is preferably approximately 15 to approximately 30 nucleotides in length, more preferably approximately 18 nucleotides in length.
  • the actual nucleotide sequence of the probe will depend on the location of the mutation in the gene for type I collagen.
  • the probe will contain the mutation with normal flanking nucleotides upstream and downstream of the mutation and is synthesized in the sense direction of the gene.
  • the nucleotide sequence of the probe is complementary to the sequence of the corresponding DNA it is designed to detect.
  • a second probe having the standard or normal base sequence for the corresponding region is also synthesized using standard techniques.
  • the second probe is preferably approximately 15 to approximately 30 nucleotides in length, more preferably approximately 18 nucleotides in length and the same length as the first probe.
  • the second probe is also complementary to the corresponding DNA sequence it is designed to detect.
  • Both probes may then be labeled with a detectable label, preferably a radiolabel such as P.
  • the probes may be labeled with P using standard methods, such as ATP labeled with P on the 8 (gamma) position and T A polynucleotide kinase according to the method of Maniatis et al. , supra.
  • Non-radiolabeled probes that contain biotinylated nucleotides introduced during the oligonucleotide synthesis may also be used. Detection of th biotinylated nucleotides may be accomplished by streptavidin and antibody-linked enzymes that generate a color reaction, such as that in the Genius system (Boehringer Mannheim
  • a hybridization assay indicates the mutation in the pro ⁇ l(I) or pro ⁇ 2(I) gene is present in the DNA of the family member
  • the diagnosis can be verified by sequencing the region of the gene suspected of containing the mutation using standard DNA sequencing techniques.
  • the DNA of the family member can be tested with or without prior amplification of the portion of the gene suspected of containing the mutation. If the family member' DNA is to be amplified prior to hybridization, primers for the polymerase chain reaction can be selected and synthesize using conventional techniques from base sequences flanking the region of the mutation, care being taken that there is n overlap of the sequence of the probe and the primers.
  • the particular sequence of the primers will depend on the location of the mutation of the pro ⁇ l(I) or pro ⁇ 2(I) genes i the first family member. Reference is made to the sequence of the pro ⁇ l(I) or pro ⁇ 2(I) genes shown herein for the sequence of the primer.
  • a probe containing th mutated base 100 could be selected to span bases 90 to 108.
  • Primers would then be selected to span non-overlapping bases outside this area.
  • the sequences of the primers would be selected to correspond to the base sequence of the pro ⁇ l(I) gene in the selected areas.
  • the particular sequence can be determined from the sequence shown herein.
  • Primers are preferably from approximately 20 to approximately 50 nucleotides in length, more preferably about 35 to 40 nucleotides in length.
  • the polymerase chain reaction is performed according to U.S. Pat. Nos. 4,683,195 or 4,683,202 or commercially available kits (Cetus Corporation, Emeryville, CA) .

Abstract

Methods of determining genetic predisposition for post-menopausal osteoporosis in mammals are disclosed. The base sequence of the two genes for type I procollagen of the person tested is compared to the sequence of standard genes for type I procollagen (COL1A1 and COL2A1). Also, the base sequences of other genes for structural proteins found in bone of the person tested are compared to the standard genes for the proteins. Differences in the base sequence from the test DNA indicate an increased likelihood that the person will develop post-menopausal osteoporosis. Family members of the mammal tested can also be tested to determine if they also have a genetic predisposition to osteoporosis by testing for the presence of mutation in the type I procollagen genes of family members.

Description

METHODS OF DETECTING A GENETIC PREDISPOSITION FOR OSTEOPOROSIS
INTRODUCTION
Research for this invention was supported in part by the National Institutes of Health Grant AR-38188. The United States government may have certain rights in the invention. FIELD OF THE INVENTION
The present invention relates to the field of methods for detecting genetic diseases and more particularly to the field of methods for detecting genetic diseases linke to anomalies of genes for collagens and other structural proteins found in bone. BACKGROUND OF THE INVENTION Post-menopausal osteoporosis is an important cause of serious disability. The disease is usually defined as a condition in which there is marked decrease in bone mass (osteopenia) associated with one or more fractures from relatively minor trauma. Two forms of the disease have been described: Type I or post-menopausal osteoporosis that primarily affects women within 10 to 15 years after menopause, and type II or age-related osteoporosis that occurs in both men and women over the age of 70. Both types of osteoporosis are probably determined, in part, by insufficient accumulation of skeletal mass in young adulthood. Normally, bone mass increases until the age of 3 and then declines. When bone density drops below a threshol level, fractures occur following minor trauma. Type I osteoporosis is apparently triggered by the transient acceleration of bone loss that occurs with the fall in estrogen levels of women at menopause. It is believed that the disease is familial.
The bone loss that characterizes osteoporosis cannot be effectively reversed by any known therapies. However, the rate of bone loss can be decreased by administration of estrogens and several other agents. It is generally not advisable to administer estrogens and related agents in large doses that are needed for their effectiveness to all individuals because the therapies have side effects such as feminizing effects in men and increasing the chances of cervical and breast cancer in women. However, if women predisposed to type I osteoporosis can be identified early, it could be justifiable to accept the risk of administering estrogens or related agents and to monitor carefully for the early development of cancers so as to avoid the devastating effects of the osteoporosis itself. Bone is a complex structure whose strength and resistance to fractures depends on a number of factors. A major source of the strength of bone are fibrils of type I collagen that form a scaffold on which the mineral of bone is deposited. The proper deposition of mineral on the collagen fibrils of bone probably depends other structural proteins such as osteopontin that bind to the collagen fibrils and participate in the mineralization process.
Type I collagen is a member of a family of fibrillar collagens and accounts for 80 to 90% of the protein found in bone. It is also found in large amounts in tissues such as skin, ligaments and tendons. Type I collagen is first synthesized as a precursor known as type I procollagen that is formed from two identical proαl(I) chains and one slightly different proα2(I) chain. Each chain contains three separate domains. The N-propeptide domain at one end of a proα chain contains a globular subdomain, a short triple-helical subdomain and another short subdomain that forms part of the cleavage site to the removal of the N-propeptide. The C-propeptide domain at the other end of a proα chain is entirely globular. In the large central regio of the procollagen molecule, the two proαl(l) and proα2(I) chains are coiled into a left-handed helix and the three helical chains are twisted around each other into a right-handed superhelix to form a triple-helical structure that is characteristic of all collagens. The major triple-helical region of each proα chain is called the α-chain domain. Each a chain contains about 1,000 amino aci residues, and with the exception of a short seguence at the end of the chains, every third amino acid is glycine. Therefore, the molecular formula of an α chain can be represented as (-Gly-X-Y-) , where X- and Y- positions denote amino acids other than glycine. The presence of glycine, th smallest amino acid, in every third position is critical, since the amino acid in this position fits into a restricted space in which the three chains come together in the center of the triple helix. The X- and Y- positions are frequently occupied by proline and 4-hydroxyproline, respectively. The biosynthesis of the procollagen molecule involves a large number of post-translational modifications that require at least eight specific enzymes and several non-specific enzymes. In total, over 100 amino acids in each a chain are modified. After the protein is assembled, it is secreted from cells and the procollagen converted to collagen by cleavage of the N-propeptide with one enzyme and the cleavag of the C-propeptide to the second enzyme. With the cleavage of procollagen to collagen, the solubility of the protein drops dramatically and the collagen self-assembles into collagen fibrils. In many tissues the type I collagen fibrils are found in association with other types of collagens and other components of the extracellular matrix. The proαl(I) chain of type I procollagen is synthesized on one gene (COLlAl) found in chromosome 17. The proα2(I) chain of type I procollagen is synthesized on the second gene (C0L2A1) found in chromosome 7. Mutations in th genes that alter the structure of the proαl(I) or proα2(Σ) chains or that decrease expression of the proαl(I) or proα2(I) have been shown to cause osteogenesis imperfecta, a genetic disease of children characterized by brittleness of bone. Many but not all children with osteogenesis imperfect also have blueness of the sclerae of the eyes, poor dentitio and thin skin because of a decrease in the amount of type I collagen or because of the formation of abnormal type I collagen fibrils. The brittleness of bone seen in osteogenesis imperfecta is usually apparent early in childhood because the patients develop many fractures for minor trauma. Many of the patients with mild forms of the disease become fracture-free after the growth spurt of puberty but then develop a marked tendency to fracture later in life.
Mutations in the gene for type II procollagen have been shown to cause genetic disorders (chondrodysplasias) of cartilage, a tissue that is rich in this protein. Mutations in the gene for type III procollagen have been shown to caus the type IV variant of Ehlers-Danlos syndrome that is characterized by thinness and other abnormalities of skin together with sudden rupture of the aorta and other hollow organs that are rich in type III collagen. Mutations in the gene for type III procollagen have recently been shown to also cause familial vascular aneurysms in families without any evidence of a genetic disease such as the Ehlers-Danlos syndrome or the Marfan syndrome. Mutations in one of the genes for type IV collagen have been shown to cause the Alport syndrome characterized by kidney disease and hearing loss. Mutations in the gene for type VII collagen cause a severe form of epidermolysis bullosa, a disease characterize by rupture and blistering and scarring of the skin from mino trauma. The structure of collagen in heritable collagen diseases are reviewed in Prockop and Kivirikko (1984) , New England Journal of Medicine, 311:376-386; Prockop (1985),
Journal of Clinical Investigation , 75:783-787; Prockop (1986) Hospital Practice , February 15, 1986; Prockop, Journal of Biological Chemistry, 265:15349-5352 (1990); and Kuivanie i et al. (1991) FASEB Journal , 5:2052-2060.
Less is known about the role of other proteins found in bone that contribute to the proper formation and, therefore, normal structure of the tissue. A number of proteins have been suggested as candidates for proteins that bind to collagen fibrils in bone and then assist in the correct deposition of calcium and phosphate. The proteins suggested as candidates for this role include osteonectin, bone Gla-protein, and osteopontin. The gene for human osteopontin has recently been cloned and located on chromosome 4 on the long arm near the centromere; Young et al. (1990) Genomicε , 1, 491-502. No human disease has yet been shown to be caused by mutations in genes for structural proteins of bone other than the two genes for type I procollagen. However, many forms of osteoporosis have been shown to be inherited and the inheritance is usually in a Mendelian dominant manner (see R. L. Riggs and L. J. Melton, 1986, N. Engl . J. Med. , 314:1676-1686; and Seeman et al., 1989, N. Engl . J. Med. , 320:554-558). Genetic diseases that are dominantly inherited are usually caused by mutations in genes for structural proteins. Therefore, forms of osteoporosis not caused by mutations in one of the two genes for type I procollagen are likely to be caused by mutations in genes that code for the other structural proteins found i bone. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic diagram of the 3'-half of the human proαl(I) gene. Figures 2A and 2B show the nucleotide sequences of the human COLlAl gene extending from intron 25 to exon 52. Capital letters indicate terminal codons of exon sequences o five ambiguous bases in introns. The alternative bases in some cloned PCR products were G/T at position +185 in intron 46; A/C at position +291 in intron 46; T/C at position +112 in intron 49; A/G at position +126 in intron 49; and C/T at position +179 in intron 50. SUMMARY OF THE INVENTION
The present invention provides methods of screening persons to determine whether those persons have an increased likelihood of developing osteoporosis. Persons not otherwis known to have a connective tissue disease are screened to detect mutations in the two genes for type I procollagen tha indicate increased likelihood of developing osteoporosis. I the methods of the invention, a tissue sample is provided an the nucleotide base sequence of at least a portion of the DN for the proαl(I) and the proα2(I) chain derived from the tissue sample is determined. The DNA for the chains can be genomic DNA or cDNA prepared from mRNA. The nucleotide base sequence of the two genes for type I procollagen (COLlAl and C0L1A2) are then compared to the nucleotide base sequence of standard DNA sequence for the same two genes to determine differences in nucleotide bases at corresponding regions of the DNA. A difference in the base sequence of the DNA from test sample as compared with a standard sequence indicates a increased likelihood of the mammal suffering from osteoporosis.
The methods of the invention make it possible to detect mutations in the two genes for type I procollagen in persons not otherwise known to have a connective tissue disease. The methods of the invention also make it possible to diagnose collagen diseases that are reflected by mutations in the type I procollagen genes such as osteogenesis imperfecta, in persons who do not exhibit symptoms of the disease and who have not been diagnosed by physical symptoms of the disease (because symptoms are variable, also indicative of other diseases, or too mild for clinical or physical diagnosis to be made) . While some forms of osteogenesis imperfecta can be diagnosed by the presence of physical symptoms alone, the appearance of the disease in the general population may be more variable, with its presence in some persons and families remaining undetected because of the mildness or variability of the physical symptoms. In such cases, the methods of the invention may be particularly useful.
It has now been found that mutations in the genes for type I procollagen increase the likelihood of developing osteoporosis. It has also been discovered that, when present, family members have the mutation in either of the two genes in the same location. Thus, if one family member develops post-menopausal osteoporosis, other family members may be at risk for developing the osteoporosis. Asymptomati relatives of the family member can be screened to determine if they have the mutated gene and, therefore, are prone to develop osteoporosis.
Accordingly, the methods of the invention are also useful for detecting genetic familial predisposition to osteoporosis. In these methods of the invention, the location of the mutation in either the gene for the proαl(I) chain or the gene for the proα2(I) chain of a first family member known or suspected of having osteoporosis is determined. The nucleotide sequence of at least the mutated region is then compared to the corresponding region in the same gene of a second family member, whereby the presence in the second family member of the mutated region indicates an increased likelihood of osteoporosis in the second family member. If the development of osteoporosis or a predisposition to osteoporosis can be detected early, effective treatment with estrogens or related agents can be carried out so that the rate of bone loss is slowed before i reaches a threshold for fractures. Treatments with estrogen or related agents usually have side effect such as increased risk of cervical or breast cancer. However, patients can be carefully monitored for the early appearance of these cancer and so the effects of the cancers themselves can largely be prevented. Also, persons who are members of families predisposed to severe osteoporosis can seek appropriate genetic counseling. Although mutations of collagen genes have been demonstrated for diseases such as osteogenesis imperfecta, Ehlers-Danlos syndrome, and related disorders, it was heretofore unknown that mutations in the genes for type I procollagen are linked to osteoporosis in the absence of any other evidence of a connective tissue disease or syndrome. Type I collagen is the most abundant components o bone and probably the single most important contributor to the physical strength of the bone and its ability to withstand pressure. Mutations in the two genes for type I procollagen involving a single base or amino acid substitution may have very subtle effects on the physical properties of the procollagen. These mutations may well produce changes in the size or other features of collagen fibrils that make the bone less able to withstand the stresses of pressures in exercise and trauma over a number o years. Mutations in the genes may also decrease the expression of the gene in the sense of decreasing the amount of protein that is synthesized. As a result, the bones have an increased likelihood of structural failure resulting in osteoporosis and easy fracturing.
The evidence that mutations in the genes for type I procollagen can cause osteoporosis suggests that mutations in other genes that are required for the normal strength of bone can also cause osteoporosis. Therefore, one part of the present invention is to detect mutations in genes for other structural proteins of bone that cause osteoporosis.
DETAILED DESCRIPTION OF THE INVENTION The methods of the invention have two stages. In the first stage, a person who has developed osteoporosis or is suspected of having or developing osteoporosis is tested to determine if he or she has a mutation in the DNA sequence of the proαl(I) or proα2(I) gene (COLlAl or COL1A2) . The comparison is done by comparing the corresponding regions of the DNA sequence from the person tested with a standard DNA sequence of the same genes. The DNA sequence of the two genes from the person tested can be genomic DNA or cDNA prepared from RNA derived from a tissue sample taken from t person. A standard DNA sequence for the genes can be obtained by reference of known sequences or to those set forth herein in Figures 1 and 2 and listed in the sequence listing. A difference in the base sequence of the DNA from the person tested as compared with the standard sequence indicates an increased likelihood of the mammal suffering from osteoporosis. For the first family member tested, all or a substantial portion of DNA coding for the two genes is sequenced and compared to a standard sequence. Sequencing o the first family member's DNA may be done by conventional DN sequencing techniques such as in Example 1.
Once the location of the gene mutation is known, i can be looked for in members of the first person's family. For each genetically predisposed individual family member, the mutation in the gene is expected to appear in th same position in the proαl(I) or proα2(I) gene. For example in Family A, the genetic mutation may be at position 30; and for Family B the genetic mutation may be at position 550. Testing the family members can be done by comparing corresponding regions of the family member's genes and the mutation to determine if the mutation is present in the family members in the second stage of the methods of the invention. As used herein, the term family members means persons genetically related to one another in any degree, such as parent-child, siblings, cousins, etc.
In the practice of the methods of the invention, DNA is extracted from a test sample of cells of the family members to be tested by conventional techniques that involve lysis of the cells with sodium dodecyl sulfate (SDS) and digestion of proteins with proteinase K followed by extraction with phenol and chloroform, and ethanol precipitation as described in Maniatis et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982) , pp. 280-281. A sample of cells can be taken from many types of tissues, such as a piece of skin, a sample of blood, or by scraping of the interior of the mouth. Alternatively, mRNA can be extracted from the test sample and cDNA synthesized with reverse transcriptase, and the resulting cDNA used for analysis. The DNA derived from the cells of the test sample is then analyzed to determine whether or not the proαl(I) or proα2(I) gene for type I procollagen contains a mutation. I a mutation is found in either of the two genes, a rapid test can be devised to test other members of the patients' families to determine whether or not they have the same mutation. Family members who have the mutation can then be followed by techniques such as x-ray or photon absorption fo development of the osteopenia that is an early sign of osteoporosis and treated with estrogen or related agents so as to decrease the normal rate of bone loss with age. Most importantly, the treatment can be initiated before there is evidence of a marked decrease in bone mass.
Although the methods of the invention have been demonstrated in the first instance in humans, they are expected to be useful in other mammalian species, particularly commercially important species and laboratory animal species used in models of human disease.
Some of the sequences for the normal proαl(I) and proα2(I) genes can be found in literature. For example, in Bernard et al. (1983) Biochemistry, 22:5213-5223;
Bernard et al. (1983) Biochemistry, 22:1139-1145; Chu et al. (1984) Nature, 310:337-340; Barsh et al. (1984) Journal of Biological Chemistry, 259:14906-14913; Chu et al. (1985) Journal of Biological Chemistry, 260:2315-2320; Chu et al. (1985) Journal of Biological Chemistry, 260:691-694; Barsh e al. (1985) Proceedings of the National Academy of Sciences, 82:2870-2874; Ramirez et al. (1985) Annals of the New York Academy of Science, 460:117-129; D'Allesio et al. (1988) Gene, 67:105-115; Tromp et al. (1988) Biochemistry Journal , 253:929-922; Kuivaniemi (1988) Biochemistry Journal ,
252:633-640. However, the sequences for introns 25 to 51 presented in Figure 2 are part of the present invention. These sequences for introns 25 to 51 are important for developing oligonucleotide primers to amplify and sequence genomic DNA from patients suspected of having mutations in the gene for proαl(I) chain of type I procollagen that cause osteoporosis.
Oligonucleotide primers for amplifying and sequencing the cDNA for the human proαl(I) chain of type I procollagen has been published by Labhard and Hollister (1990) Matrix, 10:124-130. Primers for amplifying the cDNA for the human proαl(I) chain are presented in Table 1 and are part of the present invention.
The same experimental techniques for detecting mutations in the two genes for type I procollagen can readily be applied to detecting mutations in genes coding for other structural proteins found in bone. For example, the nucleotide sequences of the osteopontin gene can be used to design oligonucleotide primers to amplify genomic DNA or cDNA for the gene with the polymerase chain reaction. The products obtained from the polymerase chain reaction can then be used to define the base sequences of the gene itself or the cDNA. Therefore, mutations in the genes for these other structural proteins that cause osteoporosis can be detected in the same manner as mutations in the two genes for type I procollagen.
EXAMPLES
Example 1
Determination of the Presence of a Mutation in the Proαl(I) or the Proo2(I) Gene. Complementary DNA (cDNA) covering the complete length of the mRNA for the proαl(I) and proα2(I) chain were prepared. To prepare the cDNA, total RNA (including messenger RNA) was isolated from cultured skin fibroblasts b lysis of the cells with the detergent Sarcosyl in the presence of guanidinium isothiocyanate and pelleting the RNA through cesium chloride according to the method of Maniatis et al., supra, p. 196. cDNA was synthesized from the poly(A)+RNA, using a kit purchased from BRL (Bethesda Research Laboratories, Bethesda, MD) or Pharmacia (Pharmacia-LKB, Piscataway, NJ) or reverse transcriptase and a primer specific for the proαl(I) chain such as those provided in Table 1.
TABLE 1 OLIGONUCLEOTIDE PRIMERS
Figure imgf000015_0001
a Primers were paired in the order presented, e.g., CDC27 with CDC28, CDC22 with CDC26, etc. b Underlined nucleotides indicate sequences added to provide restriction site used to clone the PCR products.
The synthesis of cDNA using reverse transcriptase and prime was performed according to the method of Maniatis et al . , (1983) supra, p. 213. Double-stranded cDNA was synthesized according to the method of Gubler and Hoffman (1983) Gene, 25:263-269 as amended by the manufacturers of the cDNA kits. Single-stranded cDNA was synthesized using reverse transcriptase followed by alkaline hydrolysis of the RNA according to Maniatis supra, pp. 214-216. The double- or single-stranded cDNA was amplified using a polymerase chain reaction kit (GeneA p DNA Amplification Reagent Kit, Perkin Elmer Cetus, Norwalk, CT) according to instructions provided by the supplier. Primers complementary to different portion of the proαl(I) or proα2(I) gene were used in the polymerase chain reaction. The primers used are listed in Table 1. Several different combinations of primers can be used to generate DNA containing mRNAs for type I procollagen. To obtain the nucleotide sequences of the DNA produced by the polymerase chain reaction, PCR was carried out with asymmetric ratios of two primers so as to generate an excess of single-stranded DNA. The single-stranded DNA was then used as template to determine the nucleotide sequence with the dideoxynucleotide chain termination method using internal primers and a sequencing kit (Sequenase; United States Biochemical Corp. , Cleveland, OH) .
In a typical experiment, the cDNA was used as template for polymerase chain reaction with primers in Table 1 at 94°C for 1.5 minutes, 56°C or 58°C for 1 minute and 74"C for 1.5 minutes. The picomolar ratio of the forward primer to the reverse primer was 20:4 and 30 cycles of amplification were performed. In a second polymerase chain reaction, 1/100 of the first polymerase chain reaction product was used as template for 20 cycles in which the picomolar ratio of the forward or reverse primers were 50:1. The final product was purified, and the volume reduced using Ultrafree MC filtration units (Millipore No. UFC3TTK00) . The purified DNA was then used directly for the dideoxynucleotide chain reaction, as described by Sanger et al. (1977) Proc. Nat. Acad. Sci. USA, 74:5463-5467, using modified T7 DNA polymerase (United States Biochemical Corp., Cleveland, OH) . Both conventional and radioactive sequencin using S-dATP autoradiography and sequencing using fluorescently labeled primers and an AB1 370A automated sequencer (Applied Biosystems International, San Francisco, CA) were used to determine the sequences.
The sequences from the first family member (proband) are compared with the standard DNA sequence of th gene such as the sequences in Figure 2. Changes in the bas sequence of tested family members indicated an increased likelihood of that person developing osteoporosis. Example 2 Isolation and Characterization of Nucleotide Sequences of Introns 25 to 51 of the Proαl(I) Gene
Published reports have established the nucleotide sequences of all of the cDNA and about half the gene for th proαl(I) chain of human type I procollagen. These sequences can be found in Bernard et al. (1983) Biochemistr 22:5213-5223; Bernard et al. (1983) Biochemistry,
22:1139-1145; Chu et al. (1984) Nature, 310:337-340; Barsh al. (1984) Journal of Biological Chemistry, 259:14906-14913; Chu et al. (1985) Journal of Biological Chemistry, 260:2315-2320; Chu et al. (1985) Journal of Biological Chemistry, 260:691-694; Barsh et al. (1985) Proceedings of the National Academy of Sciences , 82:2870-2874; Ramirez et al. (1985) Annals of the New York Academy of Science , 460:117-129; D'Allesio et al. (1988) Gene , 67:105-115; Trom et al. (1988) Biochemistry Journal , 253:929-922. These dat provide all of the nucleotides of the proαl(I) gene that co for the amino acids in the protein. They also provide all the nucleotide sequences of 25 introns of the proαl(I) gene. Here the nucleotide sequences of the remaining 26 exons not previously analyzed were defined. For analysis of sequences extending from intron 25 to exon 40, a genomic fragment of the proαl(I) gene was cloned from cultured skin fibroblasts of a proband with osteogenesis imperfecta (Tsuneyoshi et al. (1991) Journal o Biological Chemistry, 266:15608-15613). The genomic DNA wa digested with BamHI in order to generate a 5-kb fragment fr the proα:1(1) gene. The BamHI digest was separated by electrophoresis on agarose gels and fragments of 2 to 6 kb were electroeluted. The fragments were cloned in the Lambda-2AP (Stratagene) and the resulting library was screened (Maniatis, supra) with a cDNA (Hf-404) probe for th proαl(I) chain (Bernard et al. (1982) supra) . One positive clone was isolated. To sequence the clone, deletion library was prepared with exonuclease III and a commercial kit (Erase-a-base, Stratagene) . Double-stranded DNA from deletion library was used for sequencing with the dideoxynucleotide procedure (Sanger et al. (1977) PNAS 74:5463-5467) and T7 polymerase (Sequenase; U.S. Biochemicals) .
The other sequences were obtained by using primers indicated in Table 1 and genomic DNA as a template for the polymerase chain reaction. The polymerase chain reaction products were used to generate single-stranded DNA with asymmetric primers and then sequenced to the dideoxynucleotide procedure. The data provided the nucleotide sequences that are indicated in Figure 2. Example 3 Post-Menopausal osteoporosis as a Result of Mutation in the Proα2(I) of Type I Procollagen
Skin fibroblasts were examined from a 52-year old woman with post-menopausal osteoporosis or type I osteoporosis. The woman was Caucasian and was evaluated at the Mayo Clinic after she had developed acute mid-thoracic pain following a severe jolt while driving a truck. X-ray examination of the spine showed an anterior compression fracture of the ninth thoracic vertebra and generalized demineralization of the spinal column consistent with osteoporosis. Bone densitometry of the lumbar spine assayed by dual-energy X-ray absorptiometry (Hologic QDR1000) was 0.75 g/cm , a value that was in the lowest second percentile for the same age and sex (mean normal value 1.13 g/cm ) . On physical examination, the sclerae had a slightly bluish cast, the skin was not abnormally thin, and there is no hyperextensibility of joints. Routine laboratory tests were normal, including serum protein electrophoresis. The patient had a normal menopause seven years earlier. There was no history of any disease or use of drug known to be associated with osteoporosis. She had tinnitus with a slight hearing loss in both ears, but did no wear a hearing aid. Evaluation of her hearing several years prior to the vertebral fracture indicated that she had a mild high-frequency sensory-neural deficit compatible with presbyacusias but without the prominent conductive loss typical of osteogenesis imperfecta. Dentition was normal. Her height prior to the vertebral fracture was normal considering the height of her parents. She had five previou fractures. At age 7 she had a fracture of the left mid-femu in a fall down an embankment in which a heavier child fell o her. At age 8 she fractured her left radius after a fall while ice skating and she refractured the left radius one year later when she fell off a barrel. At age 30 she fractured her coccyx when she fell on a slippery floor. At age 35 she fractured a right phalanx when her finger was caught in a meat grinder. Type I procollagen synthesized by the patient's dermal fibroblasts was examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. There was delaye migration in both the αl(I) and α2(I) chains derived from th secreted type I procollagen. The difference was more apparent when the vertebrate collagenase fragments were examined. There was a delayed migration of the vertebrate collagenase fragment A (amino acid residues 1-775) but not o the B fragments (residues 776-1,014). The delayed migration was explained by post-translational over-modification of the α chains since incubation of the fibroblasts with 0.3 mM α,α dipyridyl to inhibit post-translational modification of the protein by prolyl hydroxylase and lysyl hydroxylase abolishe the difference in migration.
To find the mutation that altered the coding sequences of either the αl(I) or α2(I) chain, total RNA was prepared from the patient's fibroblasts and used to synthesize first-strand cDNA. The single-stranded cDNA was then used as a template for nine polymerase chain reactions using primers that amplified all 3,052 base pairs of codingsequence for the triple-helical domains of the proαl( and proα2(I) chain. Each polymerase chain reaction product was analyzed by a procedure in which heterozygous single-bas mutations can be detected by denaturing and renaturing the products to form heteroduplexes and then treating the heteroduplexes with a water-soluble carbodiimide (Ganguly an Prockop (1990) Nucleic Acids Research, 18:3933-3939). Analysis of the nine polymerase chain reaction products by primer extension suggested that only one contained a sequenc variation. The region of interest spanned nucleotides 1,951 to 2,813 (amino acids 516 to 803 of the triple-helical domain) of the α2(I) coding sequence. The results suggested that a sequence variation was present in the region encoding amino acid residues 660 to 667 of the α2(I) chain.
To characterize the sequence variation that gave rise to the mismatch observed with the carbodiimide technique, direct sequencing was performed on the relevant polymerase chain reaction product as well as on the three polymerase chain reaction products that spanned the remainde of the coding sequences of the α2(I) triple-helical domain from the patient. The results showed a single-base mutation that changed the codon -GGT- for glycine at position 661 of the α2(I) chain to -AGT-, a codon for serine. The polymeras chain reaction products contained both A and G, indicating that the patient was heterozygous at this position. Dideoxynucleotide sequencing of three polymerase chain reaction products spanning the remainder of the coding sequences for the α2(I) triple-helical chain did not reveal any other difference that would alter an amino acid.
To confirm the mutation, the patient's genomic DNA was used as a template for the polymerase chain reaction. The polymerase chain reaction products were then hybridized with allele-specific oligonucleotides for the normal coding sequence, or for the normal coding sequence with a single-base substitution that converted the codon for glycine-661 of the α2(I) chain to a codon for serine. Both oligonucleotides hybridized with polymerase chain reaction products prepared from the patient's genomic DNA. As expected, the oligonucleotide with a serine codon did not hybridize with polymerase chain reaction products from 50 control samples from genomic DNA.
Further experiments with allele-specific oligonucleotide hybridization with polymerase chain reaction products demonstrated the mutation was not present in the genomic DNA from the patient's 89-year old mother, although she had severe thoracic kyphosis and radiographic evidence o age-related or type II osteoporosis. DNA was not available from the patient's father who had no history o fractures and who died at the age of 72 of colon cancer. However, the mutation was found by allele-specific oligonucleotide hybridization to polymerase chain reaction products of genomic DNA from the patient's three sons, ages 24, 29, and 31. The sons had suffered one to four fractures each following trauma as adolescents. Subsequent examinatio of the three sons demonstrated that none had any evidence of osteogenesis imperfecta or related genetic diseases. All, however, had markedly reduced bone density. Example 4
Testing Other Family Members For a Mutation in the Proα1(1) Gene or Proα2(I) Gene
Once the location of the mutation in the type I procollagen gene is known, other family members can be teste to determine if they also have mutations in the same gene that predisposes them to diseases such as post- menopausal osteoporosis. Testing of other family members can readily b done by probing the family members' DNA with a nucleotide probe having a base sequence complementary to the mutation i the first family member. The DNA from other family members can be genomic DNA amplified with the polymerase chain reaction or cDNA synthesized by conventional techniques usin reverse transcriptase and mRNA templates.
A preferred test format is a nucleic acid hybridization assay such as dot or slot blot assays or Southern transfer of DNA fragments after separation on agarose gel. Other methods such as restriction endonuclease digestion of amplified DNA followed by agarose gel electrophoresis and visualization of the DNA by ethidium bromide, if the mutation has created or destroyed a restriction endonuclease recognition site.
A DNA probe (oligonucleotide) having a sequence which includes the mutation in the first family member's proαl(I) or proα2(I) genes is synthesized using standard techniques. The probe is preferably approximately 15 to approximately 30 nucleotides in length, more preferably approximately 18 nucleotides in length. The actual nucleotide sequence of the probe will depend on the location of the mutation in the gene for type I collagen. The probe will contain the mutation with normal flanking nucleotides upstream and downstream of the mutation and is synthesized in the sense direction of the gene. The nucleotide sequence of the probe is complementary to the sequence of the corresponding DNA it is designed to detect.
A second probe having the standard or normal base sequence for the corresponding region is also synthesized using standard techniques. The second probe is preferably approximately 15 to approximately 30 nucleotides in length, more preferably approximately 18 nucleotides in length and the same length as the first probe. The second probe is also complementary to the corresponding DNA sequence it is designed to detect.
Both probes may then be labeled with a detectable label, preferably a radiolabel such as P. The probes may be labeled with P using standard methods, such as ATP labeled with P on the 8 (gamma) position and TA polynucleotide kinase according to the method of Maniatis et al. , supra. Non-radiolabeled probes that contain biotinylated nucleotides introduced during the oligonucleotide synthesis may also be used. Detection of th biotinylated nucleotides may be accomplished by streptavidin and antibody-linked enzymes that generate a color reaction, such as that in the Genius system (Boehringer Mannheim
Biochemicals, Indianapolis, IN) . Other types of detectable labels may also be used.
If a hybridization assay indicates the mutation in the proαl(I) or proα2(I) gene is present in the DNA of the family member, the diagnosis can be verified by sequencing the region of the gene suspected of containing the mutation using standard DNA sequencing techniques.
The DNA of the family member can be tested with or without prior amplification of the portion of the gene suspected of containing the mutation. If the family member' DNA is to be amplified prior to hybridization, primers for the polymerase chain reaction can be selected and synthesize using conventional techniques from base sequences flanking the region of the mutation, care being taken that there is n overlap of the sequence of the probe and the primers. The particular sequence of the primers will depend on the location of the mutation of the proαl(I) or proα2(I) genes i the first family member. Reference is made to the sequence of the proαl(I) or proα2(I) genes shown herein for the sequence of the primer. For example, if the mutation in the proαl(I) collagen gene is at base 100, a probe containing th mutated base 100 could be selected to span bases 90 to 108. Primers would then be selected to span non-overlapping bases outside this area. The sequences of the primers would be selected to correspond to the base sequence of the proαl(I) gene in the selected areas. The particular sequence can be determined from the sequence shown herein. Primers are preferably from approximately 20 to approximately 50 nucleotides in length, more preferably about 35 to 40 nucleotides in length. The polymerase chain reaction is performed according to U.S. Pat. Nos. 4,683,195 or 4,683,202 or commercially available kits (Cetus Corporation, Emeryville, CA) .
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(B) COMPUTER: IBM PS/2
(C) OPERATING SYSTEM: PC-DOS
(D) SOFTWARE: WORDPERFECT 5.0 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: n/a
(B) FILING DATE: herewith
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Jane Massey Licata
(B) REGISTRATION NUMBER: 32,257
(C) REFERENCE/DOCKET NUMBER: TJU-0616 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (215) 568-3100 (B) TELEFAX: (215) 568-3439 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 880
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CCCACGAAGA GCTAGGGACA AACACACCCG AGCTCGAAGG AGTCTTGGGC 50 TCTGGGCTCA GCTGTGCCGC TGACCTGCCG TGTGGCCACT CACTCTCACT 100 TTCTGGACCT CAGCCTCCCT ATCTGTAAAA TGAAAGACTT CTCGGCGGGG 150 CACGGTGGCT CATGCCTGTA ATCCCAGCAC TTTGGGAGGC CAAGGCGGGC 200 AGACCATGAC CTCAGGAGTT TGAGACCAGT CGGGCCAACA TAGTGAAACC 250 ACGTCTCTAC TAAAAATACA AAAGATTAGC TGGGTGTGGT GGTGTGCACC 300 TGTAACCCCC AAGCTAGTCA GGAGGCTGAG GCAGGAGAAT TGCATGAACC 350 CGGGAGGTGG AGGTTGCAGT GAGCTGAGAT CACGCCATTG CACTCCAGCC 400 TGGGCAACAG TGCGAGATTC CATCTCAAAA AAAAAAAAAA AAAGAAGAAA 450 GAAAGAAAGA AAAAATGAAA CACTTCTCCA GGCCTCCATG ACCACTGCTC 500 TGTCCTTGGA ATAGTGTGTT GGTGGCCCTC CACCCCGACA CGTGGGGATA 550 GGACAGGCCT TTGATATGAT AGGCACCCCC AGTCTTGGTG GATTCTTTGA 600 GGTCCAAAAG GAGATAGCAG AGAAGAGAAA GCCCTTTGCA GTGCAGGCCA 650 CAGCGGGCAT CTAACAGGGA AAAGGCAGAG GAGCCTGGAA GGGTCTCTTG 700 GGAGGAGTGG GCTCAGAAAG GGCCCAGCAA GAAGCACCTG CAGGGGCATT 750 CCCCGGGGGC CAAACAGTCT TTTGAAAAGA AAGTCCCTTA AAAAGTCCCA 800 CTCAGAGTAA ATGAGAGCCC CAGGAGGCCC TGGCTTCTCA CTTCAGCCCC 850 CTCAACCCTA ACTCCCTTTC TCCACAGGGA 880
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 149
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
( i) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GTC GTAAGTATCT CCTTTCCATC CCTACCTCCT TCCCATTGCT 43 GCCCCGGCAC TTTCTCTCCC TGCAGGAGGG GTGCTAGAGG CCACGGTCCT 93 CAGCTGCTCG GGGCCCTCCT AACCCTGAGT TCCCCTTTGC TCTCTCCCTG 143 CAGGGT 149
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 109
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GCT GTGAGTGTCC CTGATGGGGA GATCTGGGGA GCAGAAAAGG 43 GGAGACACCC TCAGCCCCTC GTCTCCTCGG CCTCCCCGTG ACTGTAGTGT 93 TCTCTCTGTG CAGGGT 109
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 117
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CAG GTGAGGCCTC ATGGCTGTCA GGATGATGGG AGGTAGGGGT 43 AGGAAACACC TCTTTGGTCT CTTCCAGATT CTAAACCTTC CCTCCCTTCT 93 TCCCCCATTT CCCACCTACA GGGT 117
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 458
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CAG GTAAGAGGGA GCAGCCGGCC AGAGGGGTGG GAGATGCAGG 43 GAATCCAGAG GGACAGGCCC CCGCCTCCTA GCTAATCAGA CAGCCATCAA 93 CTAGAGGGAT TGAGGTTAGA CACCGGAAAG AACTTCCTCC CATGAAGGGA 143 GCAGCACAGA GGGAAGTGGG GGCTGCATGA TTGCTAGTCT GGGTGACTTC 193 TTTTAAGAGC TGCTGGAATA TGCTGTGACT TTCCCTCAAC CCTTGTATTG 243 ATAAATCTTG GTCCATAGTT TGGGGAGGGG GGAAGCCTTT GACACATCCC 293 TAGGAGGAAG AGAGGGGCTG TTTGGGATAA TCTCAATTCA GTGCTGAGAA 343 GGGGTTCCT CTCTAATCACG GCCAGACCCC AGGAGGAAGG ACCGTGCTTT 393 CCAGCAGAG TGGCCCCAGGT AGGTTTTGCT CACTGTCTGT TCCTCTCTCC 443 CTCCCCCTC AGGGT 458
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 99
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
AGA GTAAGTAGGC CTCTCGCGCT GCATCCGTCA AGGTGCGTTG 43 TACTTGGCCC TATCTCCAGA GCAGCCTTCA CATGCCCTGT CCTTCCCTTC 93 GAGGGC 99 (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 298
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
AAG GTGAGGGCAG CGTGGAAGGG GCTCTGGCAA GTGGCCCAGG 43 GACCAGGTCT CACCCCTCCT GCAGCAGGGG CTGGCGGGCC ATGACCAAAG 93 CCATGGAGAT AGGGTGTGGG GTGGGGGGAA AAGACCAGGG CAGGGGCCAC 143 ACACAGCCTG GAGTCTGGCT GTGAGTCTTT TCATCTTTTC TCAAGGCTTG 193 TCGTTGGCCT TGGAAACAAG CCTGGGAGAT ACCAAGCGGG GCTTAGGGCT 243 GTGACCACTC TTGGGGCCCC AGGTCACTCC AGTCTTCTTG GTTGTCACAT 293 AGGGT 298
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 466
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
AGA GTAAGTTCAA CCTTCCCCCT CCCCTGAGCC CTACATGGCT 43 CCCATCTCTG CCTCCTTTGA ATCTCTCAGC ATCTCTCCTC TCTCTGGATC 93 TCTCCTCTTC TCGGCTAATC CTCCCCTCTT CCCCCTTTTC TCCCCTCCTG 143 GCCTTTTTGC TGATGAATCC TCTCCCTGTG GTCCAGGCCC ATCTATCCCA 193 TGGGTTACCA TGGTGATGAG AGGTGGGGGC ATCTCCTTGG TGGAGGCTCC 243 CTTATTCATC CCGCTACACA ACTCAGGGGC CTGTTAACCT CAGTTCCACC 293 TCAGTCTCCA GGCAGGCACC CTTTTTCCTG AAAGAATCTT TGAGTCCTTG 343 GCCCAGGTGG AGGCAGGGCA GAGCTGCAGA GGGCCTCTCA GGAAACCCAG 393 ACACAAGCAG AACACTATAG GTCACCTCCT TGCCCCACAC TGGAAATCTC 443 AAGCTTCTCC ATGTCTTTAG GGT 466
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 227
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
AAG GTGACCTGGC CGCCTCCCCA CCTTCTGGCC CTAACACATA 43 GCCTCCTCAG CAGGCCTGGG CACGGTTCCG TGGGGTTGCG TTGGGAGAGC 93 AGGTCCTGCC AAACTGAGCT GTCAACCTGG GAACCTGGAG GGACCAGAAG 143 GAGGGGAGGC TCTCCTGGGG TCATCTACTA GGAGTATTCA GGGGAGGCCC 193 TGACCCTGAG CCTCTTGTCC CTTGCTCTCA GGGT 227 (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 169
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
CCC GTAAGTACAG AAGACCTGTT AAGACCCCAT ACTTGGCCCT 43 TCCCTCCCTT CACACAGCAC CCCTGGCCCT GTCTGTGCCT TCACCCCTTG 93 CCTCTCCCCT CACCGCATCC CCGCCTTCCC TCCTGTCAGA CGCATCTCTC 143 CAATCTGACT CCTTTTCTTC TAGGGA 169
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 219
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CCT GTGAGTACCA AGACCCCCAT CATTTTTCAT CACCGACTGG 43 GACCTGGGAC CTCGAGGGAC GGAATGAGGA CAAAGGCGTC CGTCCTCAGG 93 GGAGAAGGGT GGAGACGGGA TTGTTTCCCA CCCAAGCATC TTCCTGCCTC 143 CATTACTGCT CCTCCCCCAG GTAGTGGAAA CTCCTCCTCC TTCCCTCCAT 193 TCACGCCCTG CTTCCTCCCC CAGGGT 219
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 103
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
ATT GTGCCCCATT GTGAGTGGCT TGGCCTCTGT GCCCACGAGG 43 CTGGTGGGCT GGGACCCAGG ACGGGTCCAG GCTTGATGCG TCTGTGCTCT 93 CCTACAGGGT 103
(2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 132
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
CCT GTGAGTATCA CCCGCCTCTC TGTTGAGCCT CTCCCCTCTC 43 CCCAGGCAGC GGTGGCAGGT GAGGGCAGCT GGGTCGGATG AGTTGGCTGT 93 TCTCCCTCTG ACTGTTCCTA TGTTCTCTCC TTCCAGGGT 132
(2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 146
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
TCT GTAAGTCTCT GCAGCAGAGT CCACTGCTCT AGGTTGGGGG 43 TGCTGGGTGG GGGCTGCCAG AAGGATGGTG GGGCTGACTG AGGACCCAAT 93 GATGCACCAG AGCCCCCTGG AGTCTGACAG CCCCTCCTAT CCTCATCCAG 143 GGA 146
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 107
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GCT GTAAGTGCCA GCTCAGATCT CTGCAGCTCC GGAGGTGTGC 43 AGAGCTGGGG AGGGGTCCCT GTGCTGCTGT CTGGCACCTC ACCCCTGTTT 93 GCCTCCCAAA GGGT 107
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 159
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
TCT GTAAGTGCCC CCCTCACCTT GGGGGCCCTG AGAAAAACCA 43 TCACAGGACT TGGAGTGGGC GGAGCCAAGG AGAACAGATT TGGTAGAGAT 93 GACTCCAGCG GACTCAAGTC CTCCCAGACC CTATCTCTGG CCTGACTCTT 143 TCTTCTCCCT TAGGGT 159
(2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GAG GTGAGCAGTC CCCAGCCCCC ATGCCAGTAC CCTCAGCATG 43 GCCATTGTGG CCTTGCCTAA GCCCTCTTCC CCGGCTGACT CTCACTTCTC 93 TCTCTCTCTC TCTGCAGGGG 113
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 110
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
AAG GTAAGATGGC AACACTCCAT GACCACAGCC TTGTCTGCTG 43 CTTCCCTGCC CCATCCTGGC CCTTCACCCG GGGCTGACCC ATATTCCCCT 93 GCTCTCCCCG CCAGGGT 110
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 384
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
ACT GTAATAGCTG GGCTCCAGTT CCCTGTACCT GGTCAGGCCA 43 GGGACTCTTC AGGCCTCCTT AGAGGCTGGG GATGGGTGTC GGACTTCACC 93 CAGGCAGGGG GAGGAAAGGA GATCCTGCAA GATGTCAGGG CCTTAATCCA 143 AAAAACTGAG TTAAAGCTCA GCCCTAAGTC CCCTCTCCCA GACAGGACCG 193 CCTCTCCCAT GAGTTGGCCC CAGCTCCCGT NAAGATTGCA GTGGGGAGGT 243 TTCCCTGGGA GTTGGGAGAG ATGGCCACAG TGGGAAGCAG CTGAGGAGAG 293 AGAGATCCAG CAGAGGGGA GGCCTCATCCT GCAGCCCCAG CCTCAGCCTT 343 CCCTGGCCAA GAGCTCATG CTTTCCTTGCT CTCCCCAGGG T 384
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GCC GTAAGTACCC TGCTGTGTCC CCCATGCCTT CAGAACTCTA 43 CAGATGCAGA CAGTGCCCCA CTCGATGCCA ATGGAACTTC CGCCTGACAG 93 TTTGTCCCTT TCTCTCTTCT AGGGA 118
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 342
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
CCT GTAAGTATGC TCAGCCCCTC CCCAGTCCCC ATGCTGTGCT 43 GTGGGATAGG AGGGGGAGCT TCGCCTCAGT TTCCCCCTCT GGATAGTCAT 93 TCTTTCCCCT CCCTAGTGGG GACTGGGGTC TGAAGATTTG TGGGCATGTC 143 CAAGTAGCTT CTGAGAGGGT GAGGGGTACA CAGAGAGGGA TTATKGGAGA 193 GGTCTCTGCC TATGGACACC CTCGGGCTAG ATTTCCAGAA TAATGAAGGG 243 GCATGGGTTG CCACACTGCC CTTGTCTCTC CAGCCAGGCC CTCAGGCTAM 293 ATTTGACGCT CACTGGGCCT GAACTGCCTT TTTATCTGTC CTTCAGGGC 342 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 363
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
CGA GTAAGTCATG CCTTCTCTCT CCTCTTCTGA GCCCCAAGCC 43 CAGGCTCACC TCGGGACCTT GCCAGGACCC AGGCACCCTT TGCCTCTCTG 93 GAGAAGGGTT CAGGGACAGG GAGTGGGCAA AGAAAGGAAG AATCCTGAAC 143 AAACAATCTG ATCTAGCTTT GGCCTCTCTG CTCCCCAATC CGTCCTCCCC 193 TGGCTCAGCG GCTGGGAGGA GCTATGGCAT GTCCTATGGA AAGAGGCTGA 243 GGCTGGCTCT ATGAGCCGTG GGGCCAGAGC CAGCAGGGAG GGTGGTGGGC 293 CTCTCCTCCA GAGCTGGGGT TGTTCGGGCT TCTGGCAGCC TTTCTCAAAC 343 CATTTCCCCC ACTCCAGGGT 363
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97
(B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
( i) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GTT GTATGTAGCC CCTCATCCCC TCTGCTCATG GCCCTCCAGC 43 CCCCATAGCA CTTGGATGCC GGAATCCCCA CTCTCTTCCC TCTCTGTGCA 93 GGGT 97
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 148
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GTG GTGTGGGCCT GCCCTAGCCT CTCCCTCCCT CCTACTCCTG 43 CCATGCCAGG GTCCCCATGC CCATATGTGC CCCTACCATA TGGTGCTGGC 93 TGCTCCCTTT CCCTGACTCC ATCTTGCCCT GCCCTRCCAC AGGAG 148 (2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 194
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
CAG GTGCGTGAGC TGGACCTCAG AGCCAGTGTT AGGAGATGGG 43 CTAGCCCAGT GCTCAGAAGG GACATGAAGT CCTGGAGTAG GTCTCTGCTA 93 AGGGTGATGG ACAGAGCTGG GCTGGGAGGC AGGGGTCTCA GGTCCCTGCT 143 AGTGGTTCAG ACACAGGCTG CCGATGGGCA GGTGGTGCYC CTCTGATATA 193 ACGGTGCATT GGGCAGCTCT CTGAGGACCC TGGACAGGAG GCCAGCAGGA 243 CTAGAGGTTC CCGCATAGCT CACTCTTCCC TCTCTCTCCT CCCTGCAGTT 293 C 294
(2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 135
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
ACG GTGAGTGCCC AGAATCCCCA GGCAGGGCCC CACCTCTCCG 43 GCCTTGGGCT TTTTGGCCAG GCCATAGTGC CCTCTCTCCA TCACTCCCAC 93 GTGGTAATGC CCCCTCCCGT TGTCTCCGCC CCACCCCAGA GT 135 (2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GGAATTCAAG CGTGGTGTGG TCGGCCTG 28 (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: unknown
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: GGGGATCCCT CACCACGATC ACCACTCT 28 (2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GGAATTCCTT GGCCCTGCTG GCAAGAGT 28 (2) INFORMATION FOR SEQ ID NO:30: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: unknown
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: AAGGATCCCA GGCGGAAGTT CCATTGGC 28 (2) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: CCGGAATTCC TGGCCAAGAG CTCATGCT 28 (2) INFORMATION FOR SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28
(B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single
(D) TOPOLOGY: unknown
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: AAGGATCCCC TCCTATCCCA CAGCACAG 28 (2) INFORMATION FOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: ATGGATCCAT GCTGTGCTGT GGGATAGG 28 (2) INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: unknown
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: ATGATTTCCG TTGAGTCCAT CTTTGCCA 28 (2) INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: ATGGATCCTC GCGGTCGCAC TGGTGATG 28 (2) INFORMATION FOR SEQ ID NO:36: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: ATGAATTCCA GCCTTGGTTG GGGTCAAT 28 (2) INFORMATION FOR SEQ ID NO:37: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: ATGGATCCAT GTCTGGTTCG GCGAGAGC 28 (2) INFORMATION FOR SEQ ID NO:38: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: ATGAATTCTC AATCACTGTC TTGCCCCA 28

Claims

What is claimed is:
1. A method of detecting a genetic predisposition for osteoporosis in a person not otherwise known to have a genetic disease of connective tissue, comprising identifying a person suspected of having a genetic predisposition for osteoporosis; providing a tissue sample from said person; determining the nucleotide base sequence of at least a portion of DNA coding for the proαl(I) gene or proα2(I) gene for type I procollagen (COLlAl and C0L1A2) derived from said tissue sample; and determining at least one difference in nucleotide base sequence of corresponding regions of said sample DNA and a standard DNA sequence in the gene for the proαl(I) or proα2(I) chain of type I procollagen whereby a difference in the base sequence of the DNA from the test sample as compared with the standard sequence indicates an increased likelihood of said person suffering from osteoporosis.
2. A method of detecting genetic familial predisposition to osteoporosis in individuals not otherwise known to have a genetic disease of connective tissue, comprising the steps
(a) determining the location of a mutation in the proαl(I) gene or proα2(I) gene of type I procollagen of a first family member known or suspected of having osteoporosis; and
(b) determining whether the proαl(I) gene or proα2(I) gene of a second family member of the mutated region indicates an increased likelihood of osteoporosis in the second family member.
3. The method of claim 2 wherein said determining step comprises
(a) providing a tissue sample from said person; (b) amplifying at least a portion of DNA for either the proαl(I) gene or proα2(I) gene of type I procollagen in said tissue sample;
(c) comparing the nucleotide base sequence of said amplified DNA with the nucleotide base sequence of a standard DNA sequence containing the proαl(I) gene or proα2(I) gene of type I procollagen to determine at least one difference in the nucleotide bases and corresponding regions of DNA, whereby a difference in the base sequence of the DNA from a test sample is compared with the standard sequence indicates an increased likelihood of said first family member suffering from osteoporosis.
4. The method of claim 1 wherein the difference in base sequence of the DNA from the test sample results in the DNA sequence coding for a different amino acid or causing decreased expression of the gene.
5. A kit for detecting a genetic predisposition for osteoporosis in a mammal suspected of having a genetic predisposition for osteoporosis comprising: (a) primers capable of amplifying at least a portion of the proαl(I) and proα2(I) genes of type I procollagen genomic DNA in a tissue or fluid sample from the mammal; and
(b) a standard nucleic acid sequence for the proαl(I) and proα2(I) genes of type I procollagen for determining at least one difference in nucleotide base sequence of corresponding regions of the sample DNA, whereby a difference in the base sequence of the DNA from the sample as compared with the standard sequence indicates an increased likelihood of said mammal suffering a vascular aneurysm.
6. The kit of claim 5 wherein the standard nucleic acid sequence is a DNA sequence.
7. The kit of claim 5 wherein the primers are capable of amplifying substantially all the regions of the proαl(I) and proα2(I) gene of genomic DNA.
8. The kit of claim 7 wherein the primers are capable of amplifying substantially all the regions of the proαl(I) and proα2(I) genes of genomic DNA as a plurality of fragments.
9. The kit of claim 8 wherein at least some of the fragments are overlapping.
10. The kit of claim 8 wherein the primers capable of amplifying the plurality of fragments are present as first reagents, each of said first reagents comprising the primers capable of amplifying one of the plurality of fragments.
11. The kit of claim 10 wherein each of the first reagents comprises two kinds of primers.
12. The kit of claim 11 further comprising a plurality of second reagents, each of said second reagents comprising two kinds of primers capable of amplifying one of the plurality of fragments.
13. The kit of claim 12 further comprising a plurality of third reagents, each of said third reagents comprising two kinds of primers capable of amplifying one of the plurality of fragments.
14. The kit of claim 5 wherein said primers are detectably labeled.
15. A kit for detecting a genetic predisposition for osteoporosis in a mammals suspected of having a genetic predisposition for osteoporosis comprising: (a) primers capable of amplifying at least a portion of the cDNA synthesized from the mRNA transcribed from the type I procollagen genomic DNA in a tissue or fluid sample from the mammal; and (b) a standard nucleic acid sequence for two genes for type I procollagen (COLlAl and C0L1A2) for determining at least one difference in nucleotide base sequence of corresponding regions of the sample cDNA, whereby a difference in the base sequence of the DNA from the sample as compared with the standard sequence indicates an increased likelihood of said mammal suffering from osteoporosis.
16. The kit of claim 15 wherein said standard nucleic acid sequence is a cDNA sequence.
17. The kit of claim 15 wherein the primers are capable of amplifying substantially the whole cDNAs for the proαl(I) and proα2(I) chains of type I procollagen.
18. The kit of claim 17 wherein the primers are capable of amplifying substantially the whole cDNAs for type I procollagen as a plurality of fragments.
19. The kit of claim 18 wherein the primers capable of amplifying the plurality of fragments are present as first reagents, each of said first reagents comprising the primers capable of amplifying one of the plurality of fragments.
20. The kit of claim 19 wherein each of the first reagents comprises two kinds of primers.
21. The kit of claim 20 further comprising a plurality of second reagents, each of said second reagents comprising two kinds of primers capable of amplifying one of the plurality of fragments.
22. The kit of claim 21 further comprising a plurality of third reagents, each of said third reagents comprising two kinds of primers capable of amplifying one of the plurality of fragments.
23. The kit of claim 15 wherein said primers are detectably labeled.
PCT/US1992/010355 1991-12-03 1992-12-01 Methods of detecting a genetic predisposition for osteoporosis WO1993011149A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US80362891A 1991-12-03 1991-12-03
US803,628 1991-12-03

Publications (1)

Publication Number Publication Date
WO1993011149A1 true WO1993011149A1 (en) 1993-06-10

Family

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WO2001044455A2 (en) * 1999-12-15 2001-06-21 Astrazeneca Ab Antisense oligonucleotides for the inhibition of expression of type i procollagen
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997032041A1 (en) * 1996-02-29 1997-09-04 Gemini Research Ltd. Determination of collagen genotype
US5922542A (en) * 1996-02-29 1999-07-13 Gemini International Holdings Limited Diagnosis of predisposition to osteoporosis
US6558905B1 (en) 1999-08-30 2003-05-06 Interleukin Genetics, Inc. Diagnostics and therapeutics for osteoporosis
WO2001044455A2 (en) * 1999-12-15 2001-06-21 Astrazeneca Ab Antisense oligonucleotides for the inhibition of expression of type i procollagen
WO2001044455A3 (en) * 1999-12-15 2002-01-10 Astrazeneca Ab Antisense oligonucleotides for the inhibition of expression of type i procollagen
US7173122B2 (en) 1999-12-15 2007-02-06 Rahul Kumar Nath Antisense oligonucleotides to type I procollagen
US7723028B2 (en) 2000-08-30 2010-05-25 Interleukin Genetics, Inc. Diagnostics and therapeutics for osteoporosis
WO2003062465A1 (en) * 2002-01-25 2003-07-31 Universidad De Barcelona Compounds that are used to diagnose a predisposition to osteoporosis

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