WO1988002783A1 - Procede et agent de detection des structures genetiques de patients ayant une grande tendance a developper un iddm - Google Patents

Procede et agent de detection des structures genetiques de patients ayant une grande tendance a developper un iddm Download PDF

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Publication number
WO1988002783A1
WO1988002783A1 PCT/DK1987/000125 DK8700125W WO8802783A1 WO 1988002783 A1 WO1988002783 A1 WO 1988002783A1 DK 8700125 W DK8700125 W DK 8700125W WO 8802783 A1 WO8802783 A1 WO 8802783A1
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WO
WIPO (PCT)
Prior art keywords
iddm
agent
hla
dna
probe
Prior art date
Application number
PCT/DK1987/000125
Other languages
English (en)
Inventor
Birgitte Michelsen
Åke LERNMARK
Original Assignee
Nordisk Insulinlaboratorium
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 Nordisk Insulinlaboratorium filed Critical Nordisk Insulinlaboratorium
Publication of WO1988002783A1 publication Critical patent/WO1988002783A1/fr
Priority to DK325288A priority Critical patent/DK165192C/da
Priority to NO882629A priority patent/NO882629D0/no
Priority to FI882868A priority patent/FI882868A0/fi

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Classifications

    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • 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
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • a process and an agent for detection of gene structures in humans having a great tendency to develop IDDM A process and an agent for detection of gene structures in humans having a great tendency to develop IDDM.
  • IDDM Insulin-dependent Diabetes mellitus
  • the pathogenesis comprises phenomena of an autoimmune nature, including the presence of insulitis islet cell autoantibodies or organospecific autoantibodies or diseases of an autoimmune nature, cf. Gepts, .
  • athologic anatomy and the pancreas in juvenile diabetes mellitus Diabetes 14:619-633 (1965).
  • Foulis A. K. and J. A. Stewart. The pancreas in recent-onset Type 1 (insulin-dependent) diabete mellitus: insulin cintent of islets, insulitis and asso- ciated changes in exocrine acinar tissue. Diabetologia. 26:456-461 (1984).
  • IDDM Individuals having certain hereditary characteristics have a particularly great tendency to develop IDDM.
  • the hereditary factor has not been fully explained, it has been found that 95% of IDDM patients has antigens for the gene structure HLA-DR 3 and/or 4; Platz. P., B. K. Jakobsen, M. Morling, L. P. Ryder, A. Svejgaard, M. Thomsen, M. Christy, H. Kromann, J. Benn, J. Nerup and A. Green, "A genetic analysis of insulin-dependent dia- betes mellitus, Diabetologia 2J,, 108-115 (1982), and Wolf, E., K. M. Spencer and A. G.
  • the probe in question is produced from genomic DNA from lymphocytes by cleavage with BamHI restriction enzyme and isolation of a 3.7 kb fragment.
  • HLA-DQ ⁇ -chain probe is non ⁇ specific.
  • the present invention is based on the finding that a con ⁇ siderably smaller DNA sequence, which forms part of the mentioned 3.7 kb fragment, has increased specificity and is particularly useful as a probe for- tissue type deter ⁇ minations to identify individuals having a risk great of developing IDDM.
  • This DNA sequence called IVSI (166 bp), and fragments of it have not been produced or iso ⁇ lated before.
  • the invention concerns an agent for detection of gene structures which are characteristic of individuals having a tendency to develop insulin-dependent- diabetes mellitus (IDDM), consisting of or containing a DNA sequence from the HLA-DQ ⁇ -chain gene, and the agent is charac ⁇ terized in that the DNA sequence comprises 154 bp from intron 1 (IVSI) and the adjoining first 12 bp of exon 2 or a reactive fragment thereof.
  • IDDM insulin-dependent- diabetes mellitus
  • the invention also concerns a process for producing the agent, said process being characterized by hybridizing chromosomes from a human cell with the agent of claim
  • the present probe hybridizes with the HLA-DQ ⁇ -chain gene, which can be detected by known tech ⁇ niques for tissue type determination.
  • DNA is isolated from nucleated blood cells from the individual in question, and they .are hybridized with an IVSI (166 bp) probe.
  • Hybridization is usually effected by usual blotting technique, using labelling of the IVSI probe with radioactive isotopes for detection of positive reaction.
  • the HLA-DR4/7 daughter also has the BamHI 3.7 kb fragment, which is therefore present on the HLA-DR4 containing chro ⁇ mosome in the mother.
  • Mononuclear cells were obtained from 30 ml of blood by Ficoll-Hypaque gradient centrifu ⁇ gation, and DNA was extracted as described below. About 50 ,ug of DNA were digested with BamHI and electrophoresed in 1% agarose gel together with suitable molecular weight markers.- The region 3.4 to 3.8 kb was cut off from the gel, and the DNA fragments were recovered by electroelu- tion. This fraction of the fragment was ligated into the BamHI site in pUC8 and used for transforming E. coli JM
  • Recombinant plasmids were identified as those giving white colonies on LB plates with 20 ,ug/ml of both IPTG and BCIG, and they were screened for HLA-DQ related se ⁇ quences by in situ hybridization on nitro cellulose fil- ters with nick translated DQ ⁇ -cDNA probe, cf. the above- mentioned literature.
  • Mononuclear cells obtained from 10 ml blood by Ficoll- Hypaque gradient centrifugation, were digested overnight at 37 °C in 0.02% proteinase K and 1% weight/volume of sodium dodecyl sulfate (SDS) in 10 mM Trsi-HCl to buffer (pH 7.4) containing 1 mM EDTA (TE buffer). After phenol and chloroform extractions, DNA was precipitated with ethanol and resuspended in TE buffer. Genomic blots
  • lymphocyte DNA 10 to 20 ,ug of lymphocyte DNA were digested with the restriction enzyme BamHI (Boehringer Mannheim, DE), elec- trophoresed overnight at 40 V in 1% flat agarose gels and then transferred to "HybondN" nylon membranes (Amers- ham, Buckingshire, GB) in a known manner.
  • BamHI Boehringer Mannheim, DE
  • elec- trophoresed overnight at 40 V in 1% flat agarose gels and then transferred to "HybondN" nylon membranes (Amers- ham, Buckingshire, GB) in a known manner.
  • Filters having a DQ ⁇ -cDNA probe were prehybridized for 2 to 16 hours in 50% formamide, S x SSC, 5 x Denhart's solution, 50 mM Na personallyP0. (ph 6.5) and 0.5 mg/ml denatured salmon sperm DNA. Hybridization was performed overnight in 50% formamide, 1 x Denhart's solution, 20 mM Na-,P0. (pH 6.5), 10% dextran sulfate, 0.2 mg/ml denatured salmon sperm DNA and 10 dpm/ml denatured probe. The washing stringency was 0.1 x SSC at 55 °C.
  • the DQ ⁇ cDNA probe was derived from the plasmid pll- ⁇ l, cf. Larhammer, D., L. Schenning, K. Gustafsson, K. Wiman, L. Claesson, L. Rask, and P. A. Peterson, "Complete amino acid sequence of an HLR-DR antigenlinker ⁇ chain as pre ⁇ dicted from the nucleotide sequence: Similarities with immunoglobins and HLA-A, -B, and -C antigens.” 'Proc. Natl . Acad. Sci. USA 79:3687-3691 (1982), by digestion with Pstl and EcoRI, and then the 800 bp fragment was eluted from agarose melting at a low temperature ("BioRad", CA).
  • a subclone of the BamHI 3.7 kb fragment cloned in pBR322 was used for constructing the IVSI probe.
  • the recombinant plasmid was digested with RSAl to provide a fragment which contains 154 base pairs from the intron and 12 base pairs from the other exon, which codes for the first region of the HLA-DQ ⁇ -chain as well as the part of pBR322 dis- posed from the BamHI site in position 375 to the RSAl site in position 2281.
  • This 2072 base pair fragment was purified by agarose gel electrophoresis in low melting agarose.
  • the BamHI 3.7 kb genomic insert was subcloned in the BamHI site by pUC19, cf. Yanisk-Perron C. , J. Vieira and J. Messing "Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors". Gene 33: 103-119 (1985). This structure was then linearized with the restriction enzymes Sphl and Hindll.
  • the precipitates were collected by centrifugation for 2 mi- nutes. The supernatants were incubated for 30 minutes at 37 °C with 25 ,ug/ml RNase A followed by phenol/ chloroform extraction. Plasmid DNA was then precipitated by incubation of the samples for 10 minutes at 0 °C in 2.5 volume ethanol. After centrifugation the pellets were washed once in 80% ethanol and once in 99% ethanol, air- dried and resuspended in TE. The deletion breakpoints were determined by agarose gel electrophoresis of a suitable restriction digestion product of plasmid DNA. This analysis made it possible to decide which clones were to be charac ⁇ terized additionally by sequencing.
  • the difference in the frequency of the individual frag ⁇ ments between control and samples was judged by the Fisher exact test or the chi square test with Yate's correction.
  • the level of significance was accepted as being p less than 0.05 after correction of the p-value for the number of variable fragments observed between various individuals.
  • the relative risk (RR) was calculated from the formula-
  • the first 154 bp of the first intron ( IVS 1) and the 225 bp from the third one ( ISV 3) are also rather comparable (91-96%) with cosII-102 and DC3 ⁇ HLA-DQ ⁇ -chain genes. All four splicing joints were in accordance with the GT/AG rule.
  • BamHI 3.7 fragment represents an HLA-DQ ⁇ -chain gene, and that two BamHI sites, defining the fragment, were localized in the first and the third intermediate sequences.
  • the IVSI probe detects differences between HLA-DR3/4 healthy individuals and IDDM individuals
  • a site specific probe was constructed from the cloned DQ ⁇ 3.5 fragment.
  • the short (166 bp) IVSI probe was ex ⁇ pected to hybridize to closely related DQ ⁇ -chain genes. Since the IVSI probe moreover primarily represents non- coding sequences, which may be less preserved than their coding counterparts, a smaller degree of cross hybridiza- tion to alleles of other genes was assumed.
  • 30 HLA-DR 3/4 positive Danish individuals were tested, 13 of whom being IDDM patients and 17 healthy control indi ⁇ viduals, to find a single hybridization pattern with just 5 fragments, viz. 12, 10, 4, 3.7 and 3.2 kb.
  • the simplified restriction pattern made it possible to test whether the IVSI probe would reveal differences be ⁇ tween randomly chosen IDDM patients and healthy indivi ⁇ duals. Such.a study would allow an analysis of the strength of the possible connection between IDDM and the HLA-DQ site. It was also important to find whether the ISV1 probe would reveal a single hybridization pattern in the back ⁇ ground population to enable studies of the relative risk of developing diabetes without prior knowledge of HLA types. Accordingly, 177 healthy adult blood donors (control individuals) were compared with 113 IDDM patients.
  • IDDM patients compared with 17/117 (10%) control indivi- duals (p less than 10 -4). There were no differences between sexes in the distribution frequency for various fragments.
  • the IVSI probe is therefore seen to cleave the HLA-DR3 haplotype into subtypes having greater or smaller probability of developing IDDM. Moreover, this family study identified an IDDM patient who is HLA-DR-/7 positive, but who was shown to have the BamHI IVSI probe 4- kb fragment asso- ciated with a blank HLA-DR allele.
  • Example 1 is repeated, except that the probe is labelled with Biotin instead of 32P. After hybridization, the bio- tin is coupled to peroxidase storeptavidin, and then the probe complex is detected by reaction with 2,2' Azino-di- ( 3-ethyl-benzothiazoline sulfonate) which is a peroxidase substrate.
  • Cells from an individual are lysed.
  • the DNA is isolated and applied to a membrane filter.
  • This filter is hybridized with a synthetic DNA probe consisting of a sequence of 12 bases or more, which occur in the probe in question.
  • the synthetic probe is labelled prior to hybridization, as described in example 1 or example 2, and detection takes place autoradiographically or enzymatically.
  • Example 3 is repeated, except that the probe is one having a sequence of least 15 bases showing 80% homology or more to a sequence of at least 15 bases in the sequence in question.
  • Example 1 is repeated, except that the probe is coupled to an arbitrary DNA sequence prior to hybridization.

Abstract

Un agent, servant à la détection chez l'homme des structures génétiques qui sont caractéristiques d'individus ayant une tendance à développer des diabètes sucrés dépendant de l'insuline (IDDM), se compose d'une séquence d'ADN provenant du gène HLA-DG à la chaîne beta et comprenant 154 bp provenant de l'intron 1 (IVS1) et les douze premiers bp adjoints de l'exon 2 ou un fragment réactif dudit exon. Est également décrit un procédé servant à l'identification de ladite structure d'ADN par hybridation de chromosomes provenant de l'individu à examiner avec l'agent se trouvant dans un état marqué et à la détection de l'hybride marqué.
PCT/DK1987/000125 1986-10-16 1987-10-15 Procede et agent de detection des structures genetiques de patients ayant une grande tendance a developper un iddm WO1988002783A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
DK325288A DK165192C (da) 1986-10-16 1988-06-15 Fremgangsmaade og middel til paavisning af karakteristiske genstrukturer hos personer med stor tilboejelighed til udvikling af iddm
NO882629A NO882629D0 (no) 1986-10-16 1988-06-15 Fremgangsmaate og middel til paavisning av genstrukturer hos personer med stor tilboeyelighet til utvikling av iddm.
FI882868A FI882868A0 (fi) 1986-10-16 1988-06-15 Foerfarande och produkt foer paovisning av genstrukturer foer personer med stort boejelse foer att utveckla iddm.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK495186A DK495186D0 (da) 1986-10-16 1986-10-16 Fremgangsmaade og middel til paavisning af genstrukturer hos personer med stor tilbaejelighed til udvikling af iddm
DK4951/86 1986-10-16

Publications (1)

Publication Number Publication Date
WO1988002783A1 true WO1988002783A1 (fr) 1988-04-21

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Application Number Title Priority Date Filing Date
PCT/DK1987/000125 WO1988002783A1 (fr) 1986-10-16 1987-10-15 Procede et agent de detection des structures genetiques de patients ayant une grande tendance a developper un iddm

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EP (1) EP0415909A1 (fr)
JP (1) JPH01501566A (fr)
AU (1) AU610842B2 (fr)
DK (1) DK495186D0 (fr)
FI (1) FI882868A0 (fr)
WO (1) WO1988002783A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989004875A2 (fr) * 1987-11-17 1989-06-01 Cetus Corporation Caracterisation et detection de sequences associees a des maladies autoimmunes
WO1996003527A2 (fr) * 1994-07-21 1996-02-08 Isis Innovation Limited Procede et sonde diagnostiques
US5763591A (en) * 1996-03-25 1998-06-09 Cedars-Sinai Medical Center Polynucleic acid sequences that are functionally associated with the development of autoimmune disease

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0084796A2 (fr) * 1982-01-22 1983-08-03 Cetus Corporation Procédé pour la caractérisation de HLA et sondes de cADN pour sa mise en oeuvre
WO1983003260A1 (fr) * 1982-03-11 1983-09-29 Per Artur Peterson Procede de determination du type de tissus codes par des genes mhc
US4623619A (en) * 1982-03-03 1986-11-18 Nordisk Insulinlaboratorium Method for the determination of liability in human individuals to develop atherosclerosis
WO1986007464A1 (fr) * 1985-06-14 1986-12-18 Genetic Systems Corporation Procedes d'identification d'alleles associes a un risque accru de diabete
EP0237362A1 (fr) * 1986-03-13 1987-09-16 F. Hoffmann-La Roche Ag Procédé et nécessaire pour la détection des variations de nucléotides spécifiques et de polymorphismes génétiques dans les acides nucléiques

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0084796A2 (fr) * 1982-01-22 1983-08-03 Cetus Corporation Procédé pour la caractérisation de HLA et sondes de cADN pour sa mise en oeuvre
US4623619A (en) * 1982-03-03 1986-11-18 Nordisk Insulinlaboratorium Method for the determination of liability in human individuals to develop atherosclerosis
WO1983003260A1 (fr) * 1982-03-11 1983-09-29 Per Artur Peterson Procede de determination du type de tissus codes par des genes mhc
WO1986007464A1 (fr) * 1985-06-14 1986-12-18 Genetic Systems Corporation Procedes d'identification d'alleles associes a un risque accru de diabete
EP0237362A1 (fr) * 1986-03-13 1987-09-16 F. Hoffmann-La Roche Ag Procédé et nécessaire pour la détection des variations de nucléotides spécifiques et de polymorphismes génétiques dans les acides nucléiques

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
Biomed Biochim ACTA Vol 44, 33-36, published 1985 No 1, (B MICHELSEN) "Indentification of an HLA-DQbeta-Chain Related Genomic Sequence Associated with Insulin-Dependent Diabetes" *
Biotechnology Vol 4, 975-981, published November 1986 (H A ERLICH et al) "HLA Typing using DNA Probes" see in particular fig 38 and fig 5 *
CHEMICAL ABSTRACTS, Vol 104, 1986, Abstract 46600g, Proc Natl Acad Sci U S A 1985, 82(23), 8139-43 *
CHEMICAL ABSTRACTS, Vol 105, 1986, Abstract 147440p, Hum Immunol 1986, 17(1), 61-8 (Eng) *
Medline, NLM Accession No 86 202269, Lancet 1986 May 10;1(8489):1058-60 *
Medline, NLM Accession No 86 253068, J Exp Med 1986 Jul 1;164(1):345-50 *
Nature, Vol 322, 64-67 published 3 July 1986, (N FESTENSTEIN et al) "New HLA DNA Polymorphisms Associated with Autoimmune Diseases" *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989004875A2 (fr) * 1987-11-17 1989-06-01 Cetus Corporation Caracterisation et detection de sequences associees a des maladies autoimmunes
WO1989004875A3 (fr) * 1987-11-17 1989-06-15 Cetus Corp Caracterisation et detection de sequences associees a des maladies autoimmunes
WO1996003527A2 (fr) * 1994-07-21 1996-02-08 Isis Innovation Limited Procede et sonde diagnostiques
WO1996003527A3 (fr) * 1994-07-21 1996-03-28 Isis Innovation Procede et sonde diagnostiques
US5763591A (en) * 1996-03-25 1998-06-09 Cedars-Sinai Medical Center Polynucleic acid sequences that are functionally associated with the development of autoimmune disease

Also Published As

Publication number Publication date
JPH01501566A (ja) 1989-06-01
DK495186D0 (da) 1986-10-16
FI882868A (fi) 1988-06-15
FI882868A0 (fi) 1988-06-15
AU8155187A (en) 1988-05-06
EP0415909A1 (fr) 1991-03-13
AU610842B2 (en) 1991-05-30

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