WO2009034199A1 - Genetic informativity study - Google Patents

Genetic informativity study 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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genetic
study
informativity
use according
amplification
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PCT/ES2008/000247
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Spanish (es)
French (fr)
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Belén LLEDÓ BOSCH
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Instituto Bernabeu, S.L.
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Publication of WO2009034199A1 publication Critical patent/WO2009034199A1/en

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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

Abstract

The invention relates to the use of unique individual gametes, specifically sperm, for carrying out genetic informativity studies, such as haplotype construction analysis, for use in pre-implantation genetic diagnosis, particularly in cases in which genetic alteration occurs de novo in a family or in the absence of biological samples from affected or carrier members of the same family.

Description

ESTUDIO DE INFORMATIVIDAD GENÉTICA STUDY OF GENETIC INFORMATIVITY
La presente invención se engloba dentro del área de ciencias de Ia salud, dentro del sector de aplicación de Medicina Reproductiva, Genética y Biología Molecular, principalmente.The present invention is included within the area of health sciences, within the field of application of Reproductive Medicine, Genetics and Molecular Biology, mainly.
ESTADO DE LA TÉCNICA ANTERIORSTATE OF THE PREVIOUS TECHNIQUE
El Diagnóstico Genético Preimplantacional (DGP) es una novedosa técnica al servicio de Ia Medicina Reproductiva que permite Ia selección de embriones provenientes de ciclos de reproducción asistida libres de una determinada anomalía genética o cromosómica, antes de ser transferidos al útero materno. Actualmente, es una de las principales vías de innovación e investigación.Preimplantation Genetic Diagnosis (PGD) is a novel technique at the service of Reproductive Medicine that allows the selection of embryos from assisted reproduction cycles free of a certain genetic or chromosomal abnormality, before being transferred to the mother's womb. Currently, it is one of the main ways of innovation and research.
El DGP se ofrece a parejas portadoras o afectas de una determinada enfermedad genética o cromosomopatía, ya que se enfrentan a un importante riesgo reproductivo pudiendo elegir entre diferentes opciones: (a) diagnóstico prenatal, (b) donación de gametos, (c) adopción o (d) no tener descendencia. El DGP es una alternativa al diagnóstico prenatal evitando que Ia pareja tenga que someterse a una interrupción del embarazo en caso de que el feto se encuentre afecto.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.
La primera aplicación fue realizada por A. Handyside en 1990 consiguiendo Ia selección de embriones libres de una enfermedad genética ligada a los cromosomas sexuales. En Ia actualidad, el DGP permite el diagnóstico de cromosomopatías incluyendo el cribado de aneuploidías cromosómicas, el sexado en parejas portadoras de enfermedades ligadas a los cromosomas sexuales y anomalías cromosómicas estructurales, empleando Ia hibridación in situ fluorescente (FISH). En combinación con Ia reacción en cadena de Ia polimerasa (PCR) se puede realizar el diagnóstico de enfermedades monogénicas. De modo que se diferencian en el DGP dos grandes bloques, desde el punto de vista de Ia metodología diagnóstica y del nivel de estudio: anomalías cromosómicas y enfermedades monogénicas.The first application was made by A. Handyside in 1990 getting the selection of embryos free of a genetic disease linked to sex chromosomes. At present, 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). In combination with the polymerase chain reaction (PCR), the diagnosis of monogenic diseases can be made. Thus, 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.
La limitación principal del DGP de enfermedades monogénicas es Ia cantidad de DNA disponible para realizar el diagnóstico, ya que disponemos de una única célula para realizar el estudio. Los métodos empleados deben ser altamente sensibles y eficaces, a Ia vez que rápidos, extremando al máximo las medidas encaminadas a prevenir Ia contaminación. Entre los métodos empleados para el DGP de las enfermedades monogénicas, los fragmentos amplificados mediante PCR se pueden analizar por: digestión con enzimas de restricción, secuenciación y análisis de polimorfismos.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. Among the methods used for PGD of monogenic diseases, fragments amplified by PCR can be analyzed by: restriction enzyme digestion, sequencing and polymorphism analysis.
Los diagnósticos que dependen de Ia amplificación del ADN mediante PCR están sujetos a una serie de problemas, tales como:The diagnoses that depend on DNA amplification by PCR are subject to a series of problems, such as:
- "alíele dropout" (ADO) fenómeno que ocurre cuando solamente uno de los dos alelos presentes es amplificado, provocando errores de diagnóstico en embriones heterocigotos.- "Aliele dropout" (ADO) phenomenon that occurs when only one of the two alleles present is amplified, causing diagnostic errors in heterozygous embryos.
- Contaminación con ADN exógeno debido al gran número de ciclos de PCR necesarios para Ia amplificación de un único genoma y a Ia posibilidad de contaminación con células del cúmulo materno o de espermatozoides. De ahí Ia necesidad de testar rigurosamente los reactivos utilizados para Ia PCR, así como liberar completamente el embrión de células del cúmulo y utilizar Ia inyección intracitoplasmática del espermatozoide (ICSI) como técnica de fertilización.- Contamination with exogenous DNA due to the large number of PCR cycles necessary for the amplification of a single genome and the possibility of contamination with maternal or sperm cells. Hence the need to rigorously test the reagents used for the PCR, as well as completely free the embryo from cluster cells and use the intracytoplasmic sperm injection (ICSI) as a fertilization technique.
- Reducida o limitada eficacia de amplificación.- Reduced or limited amplification efficiency.
La utilización de polimorfismos ligados a los genes objeto del diagnóstico es Ia estrategia más eficaz y segura al evitar los posibles errores debido al ADO1 al tiempo que permite detectar los posibles casos de contaminación con ADN exógeno, que también podrían producir errores de interpretación y por tanto diagnósticos erróneos. Para incrementar Ia sensibilidad del diagnóstico se incluye más de un polimorfismo. Generalmente, se emplean dos polimorfismos que flanquean el gen o Ia mutación responsable de Ia enfermedad, Ia combinación de los mismos conforman el haplotipo asociado o ligado a Ia enfermedad.The use of 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. To increase the sensitivity of the diagnosis, more than one polymorphism is included. Generally, 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.
El primer paso previo a cualquier DGP de enfermedades monogénicas es el estudio genético del caso índice. A partir del cual se debe realizar un estudio de informatividad familiar con el que poder conocer cuales son los polimorfismos asociados a Ia enfermedad. Para ello, debe existir algún otro miembro de Ia familia vivo, afecto o portador, y así comparando miembros sanos y afectos poder establecer cual es el haplotipo de riesgo en esa familia.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.
En casos en los que Ia mutación responsable de Ia enfermedad genética ha ocurrido de novo en una familia, es decir, sólo hay un miembro afecto vivo o bien no se dispone de muestras biológicas de afectos o portadores, el estudio de polimorfismos o segregación, a pesar de ser Ia mejor estrategia para el DGP no puede ser utilizada.In cases in which the mutation responsible for the genetic disease has occurred de novo in a family, that is, there is only one living affection member or there are no biological samples of affections or carriers, the study of polymorphisms or segregation, to Despite being the best strategy for the DGP, it cannot be used.
Otro grupo de candidatos en los que se contempla Ia posibilidad de llevar a cabo un DGP son aquellas parejas en Ia que uno de sus miembros es portador de una anomalía cromosómica. Usualmente, suelen ser portadores de anomalías cromosómicas estructurales (translocaciones, grandes inversiones) aunque también serían candidatos potenciales aquellos individuos que presentan mosaicismo para anomalías cromosómicas numéricas (mosaicos para el Síndrome de Klinefelter, mosaicos 47,XYY, etc.). Una reorganización cromosómica conlleva Ia producción de una cierta proporción de gametos desequilibrados y por Io tanto a Ia obtención de embriones cromosomicamente anormales. La probabilidad de obtener embriones cromosomicamente equilibrados, desequilibrados pero viables (y por Io tanto de posibles descendientes afectos de síndromes debidos a anomalías cromosómicas) o cromosomicamente desequilibrados no viables (que suelen cursar en abortos de primer trimestre) dependerá de cada reorganización concreta.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. Usually, 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.
Los portadores de anomalías cromosómicas también suelen ser pacientes fenotipicamente normales. No obstante, en su mayoría, conocen su riesgo reproductivo ya que es frecuente que Ia reorganización cromosómica de que son portadores tenga un origen familiar. Por otra parte, no es de extrañar que sean parejas que consultan por infertilidad debido a su dificultad para conseguir un embarazo o para obtener gestaciones evolutivas. La posibilidad de llevar a cabo un DGP en estos pacientes dependerá de cada reorganización cromosómica en concreto.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.
A Io largo de los últimos años el DGP ha aumentado ampliamente su espectro de aplicación. Otros candidatos y posibilidades diagnósticas que algunos grupos incluyen en sus programas de DGP son: pacientes con edad reproductiva avanzada, en las cuales de sugiere una selección embrionaria a través de un estudio cromosómico con el objetivo de mejorar las tasas de implantación en este grupo y reducir su riesgo de transmisión de cromosomopatías a Ia descendencia; pacientes provenientes de ICSI (Inyección Citoplasmática de Espermatozoides) por factor masculino severo en los cuales se ha detectado un incremento significativo de anomalías cromosómicas en los espermatozoides; pacientes con fallos repetidos de FIV implantación en los que se sospecha de posibles anomalías cromosómicas como las causantes de los resultados negativos obtenidos; pacientes en los que se sospecha de Ia existencia de un mosaicismo gonadal e individuos de riesgo para Ia transmisión de carcinomas hereditarios.Over the past few years, the DGP has greatly increased its application spectrum. Other candidates and diagnostic possibilities that some groups include in their PGD programs are: patients with age Advanced reproductive, in which an embryonic selection is suggested through a chromosomal study with the aim of improving implantation rates in this group and reducing their risk of transmission of chromosomopathies to the offspring; patients from ICSI (Cytoplasmic Sperm Injection) by severe male factor in which a significant increase in sperm chromosomal abnormalities has been detected; patients with repeated failures of IVF implantation in which possible chromosomal abnormalities are suspected as the cause of the negative results obtained; patients in whom the existence of a gonadal mosaicism is suspected and individuals at risk for the transmission of hereditary carcinomas.
La primera limitación técnica que impone el diagnóstico preimplantacional es Ia fuente de embriones ya que las parejas candidatas a un DGP deben someterse a una FIV con los consiguientes tratamientos, desventajas y limitaciones que el procedimiento implica. Así pues, Ia tasa de éxito de Ia FIV, estimada en un 50% de forma global, representa también una importante limitación para el DGP.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. Thus, the IVF success rate, estimated at 50% globally, also represents an important limitation for the DGP.
Por otra parte, no debemos olvidar que el número y Ia calidad de los embriones obtenidos condicionarán el éxito de un diagnóstico preimplantacional.On the other hand, we must not forget that the number and quality of the embryos obtained will condition the success of a preimplantation diagnosis.
Una limitación importante de Ia aplicación de DGP a partir de embriones se debe al estadio embrionario en el cual se practica Ia biopsia, ya que ha de permitir Ia retirada de uno o dos blastómeros sin que afecte a Ia viabilidad del embrión. Por otra parte, hemos de disponer de tiempo suficiente para llevar a cabo del estudio genético y Ia transferencia en el mismo ciclo evitando, dentro de Io posible, Ia congelación de los embriones biopsiados.An important limitation of the application of PGD from embryos is due to the embryonic stage in which the biopsy is performed, since it must allow the removal of one or two blastomeres without affecting the viability of the embryo. On the other hand, we must have enough time to carry out the genetic study and the transfer in the same cycle avoiding, as far as possible, the freezing of the biopsied embryos.
La identificación del haplotipo de riesgo de una determinada enfermedad monogénica se puede establecer empleando el ADN obtenido a partir de linfocitos de sangre periférica. Como resultado de Ia amplificación por PCR se obtienen dos alelos por cada polimorfismo en caso de que se trate de un individuo heterocigoto y un único alelo en caso de ser homocigoto. Los polimorfismos que se encuentren en estado homocigoto no pueden ser empleados para realizar estudios de segregación ya que no aportan información de cromosomas independientes. En el caso de los heterocigotos claramente se diferencian dos alelos. Cada alelo proviene de un progenitor, uno materno y otro paterno, sin poder distinguirlos sin disponer de Ia historia familiar.The identification of the risk haplotype of a given monogenic disease can be established using DNA obtained from peripheral blood lymphocytes. As a result of the PCR amplification, 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. In the case of heterozygotes, 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.
Por Io tanto, de Io que se conoce en Ia técnica se deriva que es muy deseable disponer de métodos alternativos que permitan llevar a cabo estudios de informatividad genética, particularmente en aquellos casos en los que Ia mutación responsable de Ia enfermedad genética ha ocurrido de novo en una familia o bien no se dispone de muestras biológicas de afectos o portadores.Therefore, from what is known in the art it is derived that it is very desirable to have alternative methods that allow to carry out studies of genetic informativity, particularly in those cases in which the mutation responsible for the genetic disease has occurred de novo in a family or biological samples of affections or carriers are not available.
EXPLICACIÓN DE LA INVENCIÓNEXPLANATION OF THE INVENTION
Los inventores han encontrado que es posible utilizar Ia amplificación de ADN de gametos únicos individualizados para llevar a cabo un estudio de informatividad genética, e.g. polimorfismos, segregación o mutacional, en aquellos casos en los que Ia mutación responsable de Ia enfermedad genética ha ocurrido de novo en una familia o bien no se dispone de muestras biológicas de afectos o portadores.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.
Así pues, de acuerdo con un primer aspecto de Ia presente invención, ésta proporciona el uso de gametos únicos individualizados para llevar a cabo un estudio de informatividad genética.Thus, according to a first aspect of the present invention, it provides the use of single individualized gametes to carry out a study of genetic informativity.
De acuerdo con una realización de Ia presente invención, el estudio de informatividad genética se emplea para Ia identificación del haplotipo de riesgo de una determinada enfermedad monogénica. Preferentemente, el estudio de informatividad genética se emplea para el estudio de polimorfismos, segregación o mutacionales.According to an embodiment of the present invention, the study of genetic informativity is used for the identification of the risk haplotype of a certain monogenic disease. Preferably, the study of genetic informativity is used for the study of polymorphisms, segregation or mutational.
De acuerdo con otra realización particular de Ia presente invención, el estudio de informatividad genética se emplea para el propósito de selección de gametos en el contexto de fertilización in-vitro.In accordance with another particular embodiment of the present invention, the study of genetic informativity is used for the purpose of gamete selection in the context of in-vitro fertilization.
Según una realización preferida de Ia presente invención, el estudio de ¡nformatividad genética se emplea para el propósito de diagnóstico genético preimplantacional.According to a preferred embodiment of the present invention, the study The genetic information is used for the purpose of preimplantation genetic diagnosis.
Según otra realización preferida, el estudio de informatividad genética se lleva a cabo en aquellos casos en los que Ia mutación responsable de Ia enfermedad genética ha ocurrido de novo en una familia o bien no se dispone de muestras biológicas de afectos o portadores.According to another preferred embodiment, 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.
Un segundo aspecto de Ia presente invención hace referencia a un método de estudio de informatividad genética que comprende Ia amplificación del ADN de gametos únicos individualizados mediante amplificación genómica por desplazamiento múltiple (MDA).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).
En una realización del método de Ia presente invención, dicha amplificación se lleva a cabo empleando Ia ADN polimerasa del bacteriófago ph¡29. El resultado de Ia reacción se utilizará para realizar los estudios de informatividad genética, p.e. mediante Ia amplificación de los polimorfismos y de Ia mutación pudiendo establecer el haplotipo de riesgo en casos en los que no se puedan realizar estudios de informatividad familiar (casos de novo).In an embodiment of the method of the present invention, 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).
La confluencia de dos alelos en una célula somática se debe a que Ia dotación genética de los humanos es diploide. En el caso de los organismos con reproducción sexual, antes o después de Ia formación del zigoto se debe reconstituir Ia dotación del individuo para mantener Ia genética de Ia especie. En los humanos ocurre durante Ia gametogenésis en Ia que tras Ia meiosis, los gametos o células germinales poseen Ia mitad de Ia dotación genética, haploide, que permitirá tras Ia fecundación completar Ia dotación genética diploide de Ia especie. De modo, que en cada gameto encontramos independizados todos y cada uno de los alelos del genotipo del individuo.The confluence of two alleles in a somatic cell is due to the fact that the genetic endowment of humans is diploid. 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.
De acuerdo con Io descrito en Ia presente invención, es posible llevar a cabo un estudio de forma independiente de cada gameto, Io que supone una importante ventaja respecto a las técnicas empleadas hasta Ia fecha, ya que en Ia misma célula germinal es posible identificar Ia presencia o ausencia de Ia mutación, así como los polimorfismos asociados. No siendo necesario comparar los resultados obtenidos con ningún miembro de Ia familia para establecer el haplotipo de riesgo ya que al tratarse de una célula haploide por si mismos son concluyentes.According to what is described in the present invention, it is possible to carry out a study independently of each gamete, which is an important advantage over the techniques used to date, since in the same germ cell it is possible to identify the presence or absence of the mutation, as well as the associated polymorphisms. It is not necessary to compare the results obtained with any member of the family to establish the risk haplotype since being a haploid cell by themselves are conclusive.
De modo que Ia estrategia propuesta por Ia presente invención, podrá ser aplicada de forma universal a cualquier caso para evitar requerir a miembros de Ia familia en el estudio. Así mismo, cualquier estudio genético en que se necesite realizar un análisis de polimorfismos, segregación o mutacional puede ser efectuado siguiendo este procedimiento.So that the strategy proposed by the present invention, can be applied universally to any case to avoid requiring family members in the study. Likewise, any genetic study in which it is necessary to perform an analysis of polymorphisms, segregation or mutational can be carried out following this procedure.
De este modo, de acuerdo con una realización preferida de Ia presente invención, el estudio de informatividad genética se selecciona entre estudios de polimorfismos, segregación o mutacionales.Thus, in accordance with a preferred embodiment of the present invention, the study of genetic informativity is selected from polymorphism, segregation or mutational studies.
De acuerdo con una realización de Ia presente invención, el material biológico de partida para Ia obtención de los gametos únicos individualizados se selecciona entre una muestra seminal, ovocitos y corpúsculo polar de ovocitos maduros. Preferiblemente, como material biológico de partida se emplea una muestra seminal.In accordance with an embodiment of the present invention, the biological starting material for obtaining the single individualized gametes is selected from a seminal sample, oocytes and polar corpuscle of mature oocytes. Preferably, a seminal sample is used as the biological starting material.
Por Io tanto, de acuerdo con una realización particularmente preferida, Ia invención se refiere al uso de un espermatozoide como gameto único individualizado para Ia realización de estudios de informatividad genética.Therefore, according to a particularly preferred embodiment, the invention relates to the use of a sperm as a single individualized gamete for the conduct of studies of genetic informativity.
Según una realización particular de Ia presente invención, el método comprende las siguientes etapas:According to a particular embodiment of the present invention, the method comprises the following steps:
a) aislar un único gameto de una muestra; b) poner en contacto el gameto único individualizado con un tampón de lisis alcalino; c) neutralizar Ia lisis alcalina; y d) realizar Ia amplificación con Ia ADN polimerasa del bacteriófago ph¡29.a) isolate a single gamete from a sample; b) contacting the single individualized gamete with an alkaline lysis buffer; c) neutralize the alkaline lysis; and d) perform the amplification with the DNA polymerase of bacteriophage ph29.
En el contexto de Ia presente invención, por ADN polimerasa del bacteriófago ph¡29 se entiende cualquier ADN polimerasa aislada de células infectadas con fagos del tipo ph¡29, que empleen una proteína terminal para Ia iniciación de Ia replicación del ADN. Estos fagos están descritos de forma general en Salas.y col., The Bacteriophages 169, 1988.In the context of the present invention, "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.
La amplificación genómica por desplazamiento múltiple (Múltiple Displacement Amplificaron, MDA) es una técnica conocida para Ia amplificaión del genoma completo (Whole Genome Amplification, WGA) de células. Ésta técnica, MDA, se describe en US 6977148. En Ia actualidad se comercializan kits para llevar a cabo dicha técnica, p.e. Genomiphi de Amersham Biosciences, UK. Es conocida Ia limitación de esta técnica para Ia amplificación del genoma de células únicas y/o de muestras de ADN menores de 1 ng. Sin embargo, de manera sorprendente, los inventores de Ia presente invención, han encontrado que es posible Ia amplificación del ADN de un único gameto mediante ésta técnica para su empleo posterior en estudios de informatividad genéticaGenomic amplification by multiple displacement (Multiple Displacement Amplified, MDA) 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. Currently, 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. However, surprisingly, 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
La siguiente descripción general se refiere al método de Ia presente invención en caso de utilizarse una muestra seminal como material biológico de partida para Ia obtención de los gametos únicos individualizados:The following general description refers to the method of the present invention in case a seminal sample is used as the biological starting material for obtaining the single individualized gametes:
Tras lavado de Ia muestra para eliminar el plasma seminal que pueda interferir en pasos sucesivos se realizará Ia individualización de un único espermatozoide. Para ello se utilizan técnicas de micromanipulación, aspirando los espermatozoides, uno a uno mediante pipetas estándares de microinyección a 400 aumentos bajo el microscopio invertido. Los espermatozoides individualizados se introducirán en tubos para termocicladores que contienen tampón de lisis alcalino que junto con un choque térmico se conseguirá desestructurar Ia membrana plasmática con su consiguiente rotura dejando disponible el material genético del espermatozoide. Este paso no sólo permite Ia lisis del espermatozoide sino también Ia descompactación de Ia cromatina que deja accesible Ia molécula de ADN para los pasos sucesivos. La lisis alcalina debe ser neutralizada para poder realizar Ia amplificación con Ia ADN polimerasa del bacteriófago phi29 cuyo pH óptimo esta cerca de Ia neutralidad. Una vez, equilibrado el pH y disponible Ia molécula de ADN del espermatozoide se lleva a cabo Ia amplificación del ADN con una incubación a temperatura constante durante toda una noche que finaliza con Ia inactivación de Ia enzima. El producto de Ia amplificación se purificará para eliminar sales o reactivos que puedan interferir en el análisis posterior. Una vez purificado ya disponemos de gran cantidad de ADN de un solo espermatozoide que se podrá emplear para realizar los estudios genéticos descritos anteriormente: análisis de polimorfismos y estudios mutacionales.After washing the sample to eliminate the seminal plasma that may interfere in successive steps, 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. Once the pH is balanced and the sperm DNA molecule is available, 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.
A Io largo de Ia descripción y las reivindicaciones Ia palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos.Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps.
En el contexto de Ia presente invención, y tal y como se conoce en el estado de Ia técnica, el término "informatividad" se define como estudios genéticos que permiten establecer el haplotipo de riesgo de una determinada enfermedad en una familia.In the context of the present invention, and as is known in the state of the art, the term "informativity" is defined as genetic studies that allow establishing the risk haplotype of a certain disease in a family.
Para los expertos en Ia materia, otros objetos, ventajas y características de Ia invención se desprenderán en parte de Ia descripción y en parte de Ia práctica de Ia invención. Los siguientes ejemplos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de Ia presente invención.For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples are provided by way of illustration, and are not intended to be limiting of the present invention.
EXPOSICIÓN DETALLADA DE MODOS DE REALIZACIÓNDETAILED EXHIBITION OF REALIZATION MODES
Ejemplo 1. Procedimiento de manipulación y amplificación del ADN de espermatozoides únicos para estudios de informatividad genética.Example 1. Procedure for manipulation and amplification of single sperm DNA for studies of genetic informativity.
Como material biológico de partida se empleó una muestra seminal. Se realizó el lavado de Ia misma para eliminar el plasma seminal que pudiera interferir empleando PBS en proporción 1 :2 centrifugando a 1.700 rpm durante 5 minutos. Tras el centrifugado se eliminó el plasma seminal y se resuspendió en un volumen de 400 μl. En una placa de Petri para ICSI se colocaron dos gotas de 2 μl del semen lavado junto con 2 μl de PVP para conseguir ralentizar Ia movilidad de los espermatozoides. En esa misma gota se colocaron gotas 5 μl de tampón MOPS para el lavado de los espermatozoides individualizados. Se emplearon técnicas de micromanipulación, aspirando los espermatozoides, uno a uno mediante pipetas estándares de microinyección a 400 aumentos bajo el microscopio invertido. Los espermatozoides individualizados se introdujeron en tubos para termocicladores de 0.2 mi que contenían 0.5 μl tampón de lisis alcalino (200 mM NaOH, 5OmM DTT). Los tubos se incubaron a -80 0C durante 30 minutos. Y posteriormente a 65 0C durante 10 minutos. Finalmente con Ia adición deAs a biological starting material, a seminal sample was used. 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 μl. In a Petri dish for ICSI, two drops of 2 μl of the washed semen were placed together with 2 μl of PVP to slow down the mobility of the sperm. In that same drop, 5 μl 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 μl 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
0.5 μl del tampón 200 mM Tricina pH 4.95 se consiguió neutralizar Ia lisis.0.5 μl of the 200 mM Tricine buffer pH 4.95 was able to neutralize the lysis.
Los usados celulares se emplearon directamente para Ia reacción de amplificación con Ia ADN polimerasa del bacteriófago ph¡29. Se añadieron los reactivos para Ia reacción siguiendo las recomendaciones del kit comercial Genomiphi de Amersham Biosciences, UK en un volumen final de 20 μl. La reacción se incubó a 30 0C durante toda una noche. Tras Ia incubación Ia reacción se detuvo a 65 0C durante 10 minutos. El ADN amplificado se almacenó a -20 0C.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 μl. 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.
Para llevar a cabo los diferentes estudios genéticos se llevó a cabo Ia purificación del ADN amplificado empleando cualquier kit comercial de purificación de productos de PCR. Una vez purificado se dispuso de gran cantidad de ADN de un solo espermatozoide que se pudo emplear para realizar los estudios genéticos descritos anteriormente: análisis de polimorfismos y estudios mutacionales.In order to carry out the different genetic studies, 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.
Ejemplo 2. Estudio de ¡nformatividad del Síndrome de Carney Complex tipo 1.Example 2. Study of information on Carney Complex Syndrome type 1.
Dado que Ia aplicación principal del procedimiento es el DGP se expone como ejemplo Ia realización de Ia invención en un estudio de informatividad del Síndrome de Carney complex tipo 1 (CNC1 ; OMIM #160980). El síndrome de Carney complex tipo 1 es una enfermedad autosómica dominante que afecta al gen PRKAR1A localizado en el brazo largo del cromosoma17 (17q23-q24) causando neoplasias múltiples.Since the main application of the procedure is the DGP, the embodiment of the invention is presented as an example in an informativity study of Carney complex Type 1 Syndrome (CNC1; OMIM # 160980). 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.
El estudio se realizó en un varón que es portador de Ia mutaciónThe study was conducted in a male who is a carrier of the mutation
(C769T) responsable del síndrome sin antecedentes familiares de Ia enfermedad que desea tener descendencia libre del síndrome. Por ello, se propuso realizar un DGP en el que el estudio de polimorfismos no podría realizarse debido a que se trata de un caso de novo, teniendo que emplear estrategias de diagnóstico que implican un elevado porcentaje de error. El empleo del procedimiento de Ia invención nos permite poder utilizar estudios de segregación en el DGP realizando diagnósticos más seguros y fiables.(C769T) responsible for the syndrome without a family history of the disease that wishes to have offspring free of the syndrome. Therefore, it was proposed to carry out a DGP in which the study of polymorphisms could not be carried out because it is a de novo case, having to use diagnostic strategies that involve a high percentage of error. The use of the method of the invention allows us to be able to use segregation studies in the DGP making safer and more reliable diagnoses.
Se buscaron polimorfismos que estén localizados flanqueando Ia mutación. Concretamente se emplearon D17S189 y D17S1821 ((Dib, C. y col. "A comprehensive genetic map of the human genome based on 5,264 microsatellites" Nature 380 (6570), 152-154 (1996)) que permitieron establecer el haplotipo de riesgo de Ia enfermedad.Polymorphisms that are located flanking the mutation were searched. Specifically, 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
Se llevó a cabo el procedimiento de acuerdo con el ejemplo 1 , empleando una muestra seminal que tras ser lavada, una alícuota de Ia misma fue empleada para Ia individualización de los espermatozoides en tubos de termociclador que contenían el tampón de lisis, tras el choque térmico se neutralizó y se procedió a Ia amplificación y purificación del ADN de cada espermatozoide individualizado.The procedure according to example 1 was carried out, using a seminal sample that after being washed, an aliquot thereof was used for the individualization of the sperm in thermocycler tubes containing the lysis buffer, after thermal shock It was neutralized and the DNA amplification and purification of each individual sperm was carried out.
Como resultado de Ia amplificación se obtuvo ADN suficiente para poder amplificar, del mismo espermatozoide, los polimorfismos y el exón en el que se localiza Ia mutación responsable del Carney. De este modo Ia mitad de los espermatozoides fueron portadores de un haplotipo determinado que en el que no se identificó Ia mutación, mientras que Ia otra mitad fue portador de otra combinación de polimorfismos en el que si se identificó Ia mutación pudiendo establecer el haplotipo de riesgo asociado al Síndrome de Carney en este individuo. Esta información únicamente se podría haber obtenido utilizando este procedimiento y es por tanto Ia única estrategia para poder realizar el DGP del síndrome de Carney complex tipo 1 de forma totalmente fiable. As a result of the amplification, sufficient DNA was obtained to be able to amplify, from the same sperm, the polymorphisms and the exon in which the mutation responsible for Carney is located. In this way, half of the sperm were carriers of a specific haplotype in which the mutation was not identified, while the other half was the carrier of another combination of polymorphisms in which if the mutation was identified, the risk haplotype could be established. associated with Carney syndrome in this individual. This information could only have been obtained using this procedure and is therefore the only strategy to be able to perform the PGD of Carney complex type 1 syndrome in a completely reliable way.

Claims

REIVINDICACIONES
1. Uso de gametos únicos individualizados para llevar a cabo estudios de informatividad genética.1. Use of single individualized gametes to carry out studies of genetic informativity.
55
2. Uso de acuerdo con Ia reivindicación anterior 1 , caracterizado porque el estudio de ¡nformatividad genética se emplea para Ia identificación del haplotipo de riesgo de una determinada enfermedad monogénica. 02. Use according to claim 1, characterized in that the study of genetic information is used to identify the risk haplotype of a specific monogenic disease. 0
3. Uso de acuerdo con cualquiera de las reivindicaciones anteriores 1 a 2, caracterizado porque el estudio de informatividad genética se emplea para el estudio de polimorfismos, segregación o mutacionales.3. Use according to any of the preceding claims 1 to 2, characterized in that the study of genetic informativity is used for the study of polymorphisms, segregation or mutational.
4. Uso de acuerdo con cualquiera de las reivindicaciones anteriores 1 a 3,5 caracterizado porque el estudio de informatividad genética se emplea para el propósito de selección de gametos en el contexto de fertilización in-vitro.4. Use according to any of the preceding claims 1 to 3.5, characterized in that the study of genetic informativity is used for the purpose of gamete selection in the context of in-vitro fertilization.
5. Uso de acuerdo con cualquiera de las reivindicaciones anteriores 1 a 3, caracterizado porque el estudio de informatividad genética se emplea para el 0 propósito de diagnóstico genético preimplantacional.5. Use according to any of the preceding claims 1 to 3, characterized in that the study of genetic informativity is used for the purpose of preimplantation genetic diagnosis.
6. Uso de acuerdo con cualquiera de las reivindicaciones anteriores 1 a 5, caracterizado porque se emplea en aquellos casos en los que Ia mutación responsable de Ia enfermedad genética ha ocurrido de novo en una familia o 5 bien no se dispone de muestras biológicas de afectos o portadores.6. Use according to any of the preceding claims 1 to 5, characterized in that it is used in those cases in which the mutation responsible for the genetic disease has occurred de novo in a family or 5 biological samples of affections are not available or carriers.
7. Uso de acuerdo con cualquiera de las reivindicaciones anteriores 1 a 6, caracterizado porque el material biológico de partida para Ia obtención de los gametos únicos individualizados se selecciona entre una muestra seminal, o ovocitos y corpúsculo polar de ovocitos maduros.7. Use according to any of the preceding claims 1 to 6, characterized in that the biological starting material for obtaining the single individualized gametes is selected from a seminal sample, or oocytes and polar corpuscle of mature oocytes.
8. Uso de acuerdo con cualquiera de las reivindicaciones anteriores 1 a 7, caracterizado porque el gameto único individualizado es un espermatozoide.8. Use according to any of the preceding claims 1 to 7, characterized in that the single individual gamete is a sperm.
5 9. Método de estudio de informatividad genética caracterizado porque comprende Ia amplificación del ADN de gametos únicos individualizados mediante amplificación genómica por desplazamiento múltiple. 5. Method of study of genetic informativity characterized in that it comprises the amplification of the DNA of single gametes individualized by genomic amplification by multiple displacement.
10. El método de acuerdo con Ia reivindicación anterior 9, caracterizado porque Ia amplificación genómica por desplazamiento múltiple se lleva a cabo usando Ia ADN polimerasa del bacteriófago ph¡29.10. The method according to claim 9, characterized in that the genomic amplification by multiple displacement is carried out using the DNA polymerase of bacteriophage ph29.
11. El método de acuerdo con cualquiera de las reivindicaciones anteriores 9 a 10, caracterizado porque comprende las siguientes etapas:11. The method according to any of the preceding claims 9 to 10, characterized in that it comprises the following steps:
a) aislar un único gameto de una muestra; b) poner en contacto el gameto único individualizado con un tampón de lisis alcalino; c) neutralizar Ia lisis alcalina; y d) realizar Ia amplificación con Ia ADN polimerasa del bacteriófago ph¡29.a) isolate a single gamete from a sample; b) contacting the single individualized gamete with an alkaline lysis buffer; c) neutralize the alkaline lysis; and d) perform the amplification with the DNA polymerase of bacteriophage ph29.
12. El método de acuerdo con cualquiera de las reivindicaciones anteriores 9 a 11 , caracterizado porque el gameto único individualizado es un espermatozoide. 12. The method according to any of the preceding claims 9 to 11, characterized in that the single individualized gamete is a sperm.
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