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WO2009034199A1 - Étude d'informativité génétique - Google Patents

Étude d'informativité génétique Download PDF

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Publication number
WO2009034199A1
WO2009034199A1 PCT/ES2008/000247 ES2008000247W WO2009034199A1 WO 2009034199 A1 WO2009034199 A1 WO 2009034199A1 ES 2008000247 W ES2008000247 W ES 2008000247W WO 2009034199 A1 WO2009034199 A1 WO 2009034199A1
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Prior art keywords
genetic
study
informativity
use according
amplification
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PCT/ES2008/000247
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English (en)
Spanish (es)
Inventor
Belén LLEDÓ BOSCH
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INSTITUTO BERNABEU SL
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INSTITUTO BERNABEU SL
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Publication of WO2009034199A1 publication Critical patent/WO2009034199A1/fr
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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention is included within the area of health sciences, within the field of application of Reproductive Medicine, Genetics and Molecular Biology, mainly.
  • PTD Preimplantation Genetic Diagnosis
  • the DGP is offered to couples carrying or affected by a certain genetic disease or chromosomopathy, as they face a significant reproductive risk, choosing between different options: (a) prenatal diagnosis, (b) donation of gametes, (c) adoption or (d) have no offspring.
  • the DGP is an alternative to prenatal diagnosis, preventing the couple from having to undergo an interruption of pregnancy in case the fetus is affected.
  • the first application was made by A. Handyside in 1990 getting the selection of embryos free of a genetic disease linked to sex chromosomes.
  • the DGP allows the diagnosis of chromosomopathies including the screening of chromosomal aneuploidies, sexing in couples carrying diseases linked to sex chromosomes and structural chromosomal abnormalities, using fluorescent in situ hybridization (FISH).
  • FISH fluorescent in situ hybridization
  • PCR polymerase chain reaction
  • two large blocks are distinguished in the DGP, from the point of view of the diagnostic methodology and the level of study: chromosomal abnormalities and monogenic diseases.
  • the main limitation of the PGD of monogenic diseases is the amount of DNA available to make the diagnosis, since we have of a single cell to carry out the study.
  • the methods used must be highly sensitive and effective, as well as fast, maximizing the measures aimed at preventing contamination.
  • fragments amplified by PCR can be analyzed by: restriction enzyme digestion, sequencing and polymorphism analysis.
  • ADO Aliele dropout
  • polymorphisms linked to the genes object of the diagnosis is the most effective and safe strategy to avoid possible errors due to ADO 1 while allowing the detection of possible cases of contamination with exogenous DNA, which could also cause interpretation errors and Both misdiagnoses.
  • more than one polymorphism is included.
  • two polymorphisms that flank the gene or the mutation responsible for the disease are used, the combination thereof forms the haplotype associated or linked to the disease.
  • the first step before any PGD of monogenic diseases is the genetic study of the index case. From which a family informativity study should be carried out with which to know which are the polymorphisms associated with the disease. For this, there must be some other member of the family alive, affection or bearer, and thus comparing Healthy and affectionate members to establish what is the risk haplotype in that family.
  • Another group of candidates in which the possibility of carrying out a DGP is contemplated are those couples in which one of its members is a carrier of a chromosomal abnormality.
  • they are usually carriers of structural chromosomal abnormalities (translocations, large investments) although those individuals presenting mosaicism for numerical chromosomal abnormalities (mosaics for Klinefelter Syndrome, mosaics 47, XYY, etc.) would also be potential candidates.
  • a chromosomal reorganization entails the production of a certain proportion of unbalanced gametes and therefore to the obtaining of chromosomally abnormal embryos.
  • the probability of obtaining chromosomally balanced, unbalanced but viable embryos (and therefore of possible descendants affected by syndromes due to chromosomal abnormalities) or non-viable chromosomally unbalanced (which usually occur in first trimester abortions) will depend on each specific reorganization.
  • Carriers of chromosomal abnormalities are also usually phenotypically normal patients. However, for the most part, they know their reproductive risk since it is common that the chromosomal reorganization of which they are carriers has a family origin. On the other hand, it is not surprising that they are couples who consult for infertility due to their difficulty in getting pregnant or in obtaining evolutionary gestations. The possibility of carrying out a PGD in these patients will depend on each specific chromosomal reorganization.
  • the first technical limitation imposed by the preimplantation diagnosis is the source of embryos since the couples who are candidates for a DGP must undergo IVF with the consequent treatments, disadvantages and limitations that the procedure implies.
  • the IVF success rate estimated at 50% globally, also represents an important limitation for the DGP.
  • the identification of the risk haplotype of a given monogenic disease can be established using DNA obtained from peripheral blood lymphocytes.
  • two alleles are obtained for each polymorphism in the case of a heterozygous individual and a single allele if it is homozygous.
  • Polymorphisms that are in a homozygous state cannot be employees to perform segregation studies since they do not provide information on independent chromosomes.
  • two alleles clearly differ. Each allele comes from a parent, a mother and a father, without being able to distinguish them without having family history.
  • the inventors have found that it is possible to use the DNA amplification of single individualized gametes to carry out a study of genetic informativity, e.g. polymorphisms, segregation or mutational, in those cases in which the mutation responsible for the genetic disease has occurred de novo in a family or there are no biological samples of affections or carriers.
  • genetic informativity e.g. polymorphisms, segregation or mutational
  • the present invention provides the use of single individualized gametes to carry out a study of genetic informativity.
  • the study of genetic informativity is used for the identification of the risk haplotype of a certain monogenic disease.
  • the study of genetic informativity is used for the study of polymorphisms, segregation or mutational.
  • the study of genetic informativity is used for the purpose of gamete selection in the context of in-vitro fertilization.
  • the study The genetic information is used for the purpose of preimplantation genetic diagnosis.
  • the study of genetic informativity is carried out in those cases in which the mutation responsible for the genetic disease has occurred de novo in a family or there are no biological samples of affections or carriers.
  • a second aspect of the present invention refers to a method of study of genetic informativity that comprises the amplification of the DNA of single gametes individualized by genomic amplification by multiple displacement (MDA).
  • MDA multiple displacement
  • said amplification is carried out using the DNA polymerase of bacteriophage ph29.
  • the result of the reaction will be used to carry out the studies of genetic informativity, e.g. by means of the amplification of the polymorphisms and the mutation, being able to establish the risk haplotype in cases in which family informativity studies cannot be carried out (de novo cases).
  • the confluence of two alleles in a somatic cell is due to the fact that the genetic endowment of humans is diploid.
  • the endowment of the individual In the case of organisms with sexual reproduction, before or after the formation of the zygote, the endowment of the individual must be reconstituted to maintain the genetics of the species. In humans, it occurs during gametogenesis in which, after meiosis, gametes or germ cells possess half of the haploid genetic endowment, which will allow, after fertilization, to complete the diploid genetic endowment of the species. So, in each gamete we find each and every one of the alleles of the individual's genotype independent.
  • the study of genetic informativity is selected from polymorphism, segregation or mutational studies.
  • the biological starting material for obtaining the single individualized gametes is selected from a seminal sample, oocytes and polar corpuscle of mature oocytes.
  • a seminal sample is used as the biological starting material.
  • the invention relates to the use of a sperm as a single individualized gamete for the conduct of studies of genetic informativity.
  • the method comprises the following steps:
  • DNA polymerase of bacteriophage ph29 means any DNA polymerase isolated from cells infected with phage type ph29, which employ a terminal protein for Ia initiation of DNA replication. These phages are described in general in Salas. Et al., The Bacteriophages 169, 1988.
  • Genomic amplification by multiple displacement is a known technique for the amplification of the whole genome (Whole Genome Amplification, WGA) of cells.
  • This technique, MDA is described in US 6977148.
  • kits are commercialized to carry out said technique, e.g. Genomiphi from Amersham Biosciences, UK.
  • the limitation of this technique for the amplification of the genome of single cells and / or DNA samples smaller than 1 ng is known.
  • the inventors of the present invention have found that it is possible to amplify the DNA of a single gamete by this technique for later use in studies of genetic informativity
  • the individualization of a single sperm will be performed. For this, micromanipulation techniques are used, aspirating the sperm, one by one using standard microinjection pipettes at 400 magnifications under the inverted microscope.
  • the individualized sperm will be introduced into tubes for thermocyclers containing alkaline lysis buffer that together with a thermal shock will be able to deconstruct the plasma membrane with its consequent rupture leaving the genetic material of the sperm available. This step allows not only the lysis of the sperm but also the decompaction of the chromatin that leaves the DNA molecule accessible for the successive steps.
  • the alkaline lysis must be neutralized in order to perform the amplification with the DNA polymerase of the bacteriophage phi29 whose optimum pH is close to neutrality.
  • the DNA amplification is carried out with a constant temperature incubation overnight that ends with the inactivation of the enzyme.
  • the amplification product will be purified to eliminate salts or reagents that may interfere in the subsequent analysis. Once purified, we already have a large amount of DNA from a single sperm that can be used to perform the genetic studies described above: polymorphism analysis and mutational studies.
  • the term "informativity" is defined as genetic studies that allow establishing the risk haplotype of a certain disease in a family.
  • Example 1 Procedure for manipulation and amplification of single sperm DNA for studies of genetic informativity.
  • a seminal sample was used as a biological starting material. The washing of the same was carried out to eliminate the seminal plasma that could interfere using PBS in a 1: 2 ratio by centrifuging at 1,700 rpm for 5 minutes. After centrifugation the seminal plasma was removed and resuspended in a volume of 400 ⁇ l. In a Petri dish for ICSI, two drops of 2 ⁇ l of the washed semen were placed together with 2 ⁇ l of PVP to slow down the mobility of the sperm. In that same drop, 5 ⁇ l of MOPS buffer was placed to wash the individualized sperm.
  • Micromanipulation techniques were employed, aspirating the sperm, one by one using standard microinjection pipettes at 400 magnifications under the inverted microscope. Individualized sperm were introduced into 0.2 ml thermocycler tubes containing 0.5 ⁇ l alkaline lysis buffer (200 mM NaOH, 5OmM DTT). The tubes were incubated at -80 0 C for 30 minutes. And later at 65 0 C for 10 minutes. Finally with the addition of
  • the used cell phones were used directly for the amplification reaction with the DNA polymerase of bacteriophage ph29.
  • the reagents for the reaction were added following the recommendations of the commercial kit Genomiphi of Amersham Biosciences, UK in a final volume of 20 ⁇ l.
  • the reaction was incubated at 30 0 C overnight. After incubating the reaction he stopped at 65 0 C for 10 minutes.
  • the amplified DNA was stored at -20 0 C.
  • the amplification of the amplified DNA was carried out using any commercial PCR product purification kit. Once purified, a large amount of single sperm DNA was available that could be used to perform the genetic studies described above: polymorphism analysis and mutational studies.
  • Carney complex Type 1 Syndrome is an autosomal dominant disease that affects the PRKAR1A gene located on the long arm of chromosome17 (17q23-q24) causing multiple malignancies.
  • D17S189 and D17S1821 ((Dib, C. et al. "A comprehensive genetic map of the human genome based on 5,264 microsatellites” Nature 380 (6570), 152-154 (1996)) were used to establish the risk haplotype of The disease

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

Abstract

La présente invention concerne l'utilisation de gamètes uniques individualisés, concrètement des spermatozoïdes, pour la réalisation d'études d'informativité génétique, comme l'analyse de construction d'haplotypes, à mettre en oeuvre dans le diagnostic génétique préimplantatoire, en particulier dans des cas dans lesquels l'altération génétique se produit de novo dans une famille ou bien dans lesquels on ne dispose d'aucun échantillon biologique provenant de membres affectés ou porteurs de la même famille.
PCT/ES2008/000247 2007-09-11 2008-04-15 Étude d'informativité génétique Ceased WO2009034199A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES200702427A ES2315196B1 (es) 2007-09-11 2007-09-11 Estudio de informatividad genetica.
ESP200702427 2007-09-11

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WO2009034199A1 true WO2009034199A1 (fr) 2009-03-19

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1337469A (zh) * 2000-08-08 2002-02-27 华中农业大学 猪单精子微卫星dna标记分型方法
EP1330541A1 (fr) * 2000-10-23 2003-07-30 Ingeny Holding BV Procede et appareil de detection de mutation dans un fragment d'acide nucleique contenu dans un echantillon
US6977148B2 (en) 2001-10-15 2005-12-20 Qiagen Gmbh Multiple displacement amplification

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1337469A (zh) * 2000-08-08 2002-02-27 华中农业大学 猪单精子微卫星dna标记分型方法
EP1330541A1 (fr) * 2000-10-23 2003-07-30 Ingeny Holding BV Procede et appareil de detection de mutation dans un fragment d'acide nucleique contenu dans un echantillon
US6977148B2 (en) 2001-10-15 2005-12-20 Qiagen Gmbh Multiple displacement amplification

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
ALTARESCU G. ET AL.: "Single-sperm analysis for haplotype construction of de-novo paternal mutations:application to PGD for neurofibromatosis type 1", HUMAN REPRODUCTION, vol. 21, no. 8, August 2006 (2006-08-01), pages 2047 - 2051 *
ALTARESCU G. ET AL.: "Sinqle-sperm analvsis for haplotvpe construction of de novo paternal mutations: application to PGD for neurofibromatosis tvpe 1", HUMAN REPRODUCTION, vol. 21, no. 8, August 2006 (2006-08-01), pages 2047 - 2051
DATABASE WPI Week 200239, Derwent World Patents Index; AN 2002-353134 *
DIB, C.: "A comprehensive genetic map of the human genome based on 5,264 microsatellites", NATURE, vol. 380, no. 6570, 1996, pages 152 - 154
LLEDO B. ET AL.: "Preimplantation genetic diagnosis of Marfan syndrome using multiple displacement amplification", FERTILITY AND STERILITY, vol. 86, no. 4, 1 October 2006 (2006-10-01), pages 949 - 955 *
PENG AT EL.: "Whole genome amplification from single cells in preimplantation genetic diagnosis and prenatal diagnosis", EUROPEAN JOURNAL OF OBSTETRICS&GYNECOLOGY AND REPRODUCTIVE BIOLOGY, vol. 131, no. 1, 24 February 2007 (2007-02-24), pages 13 - 20 *
PENQ ET AL.: "Whole qenome amplification from sinqle cells in preimplantation qenetic diaqnosis and prenatal diaqnosis", EUROPEAN JOURNAL OF OBSTETRICS AND GYNECOLOQY AND REPRODUCTIVE BIOLOQY, vol. 131, no. 1, pages 13 - 20
SALAS, THE BACTERIOPHAGES, vol. 169, 1988
TUR-KASPA ET AL.: "Sperm DNA qenotypinq for preimplantation genetic diagnosis (PGD)", FERTILITY AND STERILITY, vol. 82, 2004, pages S254
TUR-KASPA I. ET AL.: "Sperm DNA genotyping for preimplantation genetic diagnosis(PGD)", FERTILITY AND STERILITY, vol. 82, 1 September 2004 (2004-09-01), pages S254 *

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ES2315196B1 (es) 2010-01-12
ES2315196A1 (es) 2009-03-16

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