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WO2009037337A1 - Marqueurs de transcrits spécifiques du genre, robustes et indépendants des tissus, pour la détermination moléculaire du genre - Google Patents

Marqueurs de transcrits spécifiques du genre, robustes et indépendants des tissus, pour la détermination moléculaire du genre Download PDF

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Publication number
WO2009037337A1
WO2009037337A1 PCT/EP2008/062529 EP2008062529W WO2009037337A1 WO 2009037337 A1 WO2009037337 A1 WO 2009037337A1 EP 2008062529 W EP2008062529 W EP 2008062529W WO 2009037337 A1 WO2009037337 A1 WO 2009037337A1
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Prior art keywords
linked
specific
chromosome
gender
xist
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PCT/EP2008/062529
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English (en)
Inventor
Frank Staedtler
Nicole Hartmann
Martin Letzkus
Sandrine Bongiovanni
Martin Schumacher
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Novartis Ag
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Publication of WO2009037337A1 publication Critical patent/WO2009037337A1/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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a method for predicting the gender of a mammalian subject, wherein said prediction is based on the expression profile of transcript markers that can be applied for molecular gender determination.
  • the inactivation of one of the two X-chromosomes in each somatic cell in females is important to compensate for the gene dosage compared to males possessing only one X-chromosome.
  • one of the pair of approximately 160-Mb X chromosomes is chosen to be silenced, and this silencing is then stably inherited through subsequent somatic cell divisions.
  • Recent advances have revealed many of the chromatin changes that underlie this stable silencing of an entire chromosome. The key initiator of these changes is a non-coding, functional RNA, the X (inactive)-specific transcript (XIST) (Brown et al. 1991, Penny et al. 1996).
  • XIST RNA acts in concert with other epigenetic modifications to achieve this inactivation is still poorly understood. However, a considerable portion of the X-located genes seem to escape X-inactivation (Fisher et al. 1990, Carell and Willard, 2005).
  • the human Y-chromosome was shown to contain the least number of genes of all chromosome and acts as a genetic determinant of the male characteristic features.
  • the male specific region of the Y-chromosome (MSY) was formerly also referred to as the non- recombining region of the Y-chromosome (NRY).
  • the MSY comprises 95% of the Y- chromosome and represents a mosaic of heterochromatic sequences and three classes of euchromatic sequences including the X-transposed, the X-degenerate, and the ampliconic sequences (Skaletzky et al. 2003). 156 transcription units have been identified, which include 78 protein encoding genes that collectively encode for 27 distinct proteins. While the ampliconic genes are reported to be expressed primarily in the testis, most of the genes belonging to the X-degenerate class are described to be ubiquitously expressed (Skaletzky et al. 2003).
  • an object of the present invention is to provide a comprehensive, robust and tissue independent gender-specific transcript marker set that can be applied for molecular gender determination in human peripheral blood but also in other healthy and diseased tissues.
  • the present invention provides a method for predicting the gender of a mammalian subject, wherein said prediction is based on the expression profile of transcript markers.
  • the present invention further provides a kit for the prediction of the gender of a mammalian subject comprising means to assess the expression of said transcript markers.
  • the present invention provides a comprehensive, robust and tissue independent gender-specific transcript marker set that can be applied for molecular gender determination.
  • the present invention provides for a method for predicting the gender of a mammalian subject, wherein said prediction is based on the expression profile of a group of genes comprising the ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y- linked (DDX3Y), and the Jumonji, AT rich interactive domain 1D (JARID1D), as well as the X-chromosome specific X (inactive)-specific transcript (XIST).
  • a group of genes comprising the ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y- linked (DDX3Y), and the Jumonji, AT
  • a preferred embodiment of the present invention is a method for predicting the gender of a mammalian subject, wherein said prediction is based on the expression profile of the ribosomal protein S4, Y-linked 1 (RPS4Y1) and on the expression profile of the X- chromosome specific X (inactive)-specific transcript (XIST), and wherein said prediction is based on the ratio of the XIST expression to the RPS4Y1 expression.
  • Another preferred embodiment of the present invention is a method for predicting the gender of a mammalian subject, wherein said prediction is based on the expression profile of a group of genes comprising the ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), the X-chromosome specific X (inactive)-specific transcript (XIST) and additionally the chromosome Y open reading frame 15A and 15B (CYorf15A and CYorf15B), the ubiquitin specific peptidase 9, Y-linked (fat facets-like, Drosophila) (USP9Y), the Zinc finger protein, Y-linked (ZFY), and the chromosome Y open reading frame 14 (
  • a further preferred embodiment of the present invention is a method for predicting the gender of a mammalian subject, wherein said prediction is based on the expression profile of a group of genes comprising the ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), the X-chromosome specific X (inactive)-specific transcript (XIST) and in addition the chromosome Y open reading frame 15A and 15B (CYorf15A and CYorf15B), the ubiquitin specific peptidase 9, Y-linked (fat facets-like, Drosophila) (USP9Y), the Zinc finger protein, Y-linked (ZFY), the chromosome Y open reading frame 14
  • the subject is human.
  • the present invention provides for a kit for the prediction of the gender of a mammalian subject characterised in that it comprises means to assess the expression of the ribosomal protein S4, Y-linked 1 (RPS4Y1) gene and of the X-chromosome specific X (inactive)-specific transcript (XIST).
  • a kit for the prediction of the gender of a mammalian subject characterised in that it comprises means to assess the expression of the ribosomal protein S4, Y-linked 1 (RPS4Y1) gene and of the X-chromosome specific X (inactive)-specific transcript (XIST).
  • the present invention provides for a kit for the prediction of the gender of a mammalian subject characterised in that it comprises means to assess the expression of ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y- linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), as well as the X-chromosome specific X (inactive)-specific transcript (XIST).
  • the kit comprises means to assess the expression of ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), as well as the X- chromosome specific X (inactive)-specific transcript (XIST) and in addition the chromosome Y open reading frame 15A and 15B (CYorf15A and CYorf15B), the ubiquitin specific peptidase 9, Y-linked (fat facets-like, Drosophila) (USP9Y), the Zinc finger protein, Y-linked (ZFY), and the chromosome Y open reading frame 14 (CYorf14).
  • RPS4Y1 Y-linked 1
  • EIF1AY eukaryotic translation initiation
  • the present invention provides for a comprehensive, robust and tissue independent gender-specific transcript marker set consisting of the ribosomal protein S4, Y- linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), as well as the X-chromosome specific X (inactive)-specific transcript (XIST).
  • the comprehensive, robust and tissue independent gender-specific transcript marker set consists of the ribosomal protein S4, Y- linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), the X-chromosome specific X (inactive)-specific transcript (XIST) and in addtion the chromosome Y open reading frame 15A and 15B (CYorf15A and CYorf15B), the ubiquitin specific peptidase 9, Y-linked (fat facets-like, Drosophila) (USP9Y), the Zinc finger protein, Y-linked (ZFY), and the chromosome Y open reading frame 14 (CYorf14).
  • RPS4Y1 Y- linked 1
  • EIF1AY euk
  • the comprehensive, robust and tissue independent gender-specific transcript marker set consists of the ribosomal protein S4, Y- linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), the X-chromosome specific X (inactive)-specific transcript (XIST) and in addition the chromosome Y open reading frame 15A and 15B (CYorf15A and CYorf15B), the ubiquitin specific peptidase 9, Y-linked (fat facets-like, Drosophila) (USP9Y), the Zinc finger protein, Y-linked (ZFY), the chromosome Y open reading frame 14 (CYorf14), the ubiquitously transcribed tetratricopeptide repeat gene,
  • the present invention provides for a threshold independent, ratio based gender-specific marker set consisting of the ribosomal protein S4, Y-linked 1 (RPS4Y1) and on the expression profile of the X-chromosome specific X (inactive)-specific transcript (XIST).
  • FIG. 1a Ribosomal protein S4, Y-linked 1 (RPS4Y1): 201909_at.
  • FIG. 1b Eukaryotic translation initiation factor 1A, Y-linked (EIF1AY):
  • Figure 1e X (inactive)-specific transcript (XIST): 224588_at.
  • Figure 1g X (inactive)-specific transcript (XIST): 227671_at.
  • Figure 1h X (inactive)-specific transcript (XIST): 214218_s_at.
  • Figure 1 i X (inactive)-specific transcript (XIST): 221728_x_at.
  • Marker in the context of the present invention refers to genes and gene expression products (i.e. proteins or polypeptides, mRNA) which are differentially expressed in a sample taken form subjects from one gender as compared to subjects from the other gender or another reference standard.
  • gene expression products i.e. proteins or polypeptides, mRNA
  • Marker set in the context of the present invention refers to a group of genes as described above, with a particularly high predictive power, which allows for the prediction of the gender of a subject.
  • the term “gender” refers to either the male or the female sex.
  • Proteins or polypeptides or peptides of the present invention are contemplated to include any fragments thereof, in particular, immunologically detectable fragments.
  • test samples refers to a sample from a subject obtained for the purpose of identification, diagnosis, prediction or monitoring.
  • Preferred test samples include blood, serum, plasma, lymph, urine, tear, saliva, cerebrospinal fluid, leukocyte ot tissue samples.
  • test samples would be more readily analyzed following a fractionation or purification procedure, for example separation of whole blood into serum or plasma components.
  • a gene "expression profile” as used herein refers to the differential expression of genes or protein products between the test sample and the reference.
  • the "differential expression” as used herein means the expression level of a gene or protein in the test sample compared to the reference.
  • a difference in the level of gene expression or of peptide level is a relative difference. For example it may be the difference in the level of gene expression in a sample taken from a subject having one gender (e.g. female), compared to the control subject having the other gender (e.g. male).
  • a comparison can be made between the level of gene expression in two subjects of different gender.
  • a comparison can be made to a "reference standard" known to be associated with a good outcome, such as a gene known not to be differentially expressed in the male and female gender, that is a gene with a homogenous expression.
  • the level of gene espression of the level of proteins/peptides present in a sample being tested can be either in absolute amount (e.g. ⁇ g/ml) or a relative amount (e.g. relative intensity of signals).
  • a difference is present between the two samples if the amount of gene expression is statistically significantly different from the amount of the polypeptide in the other sample. For example there is a difference in gene expression of in the level of proteins/peptides between the two samples if the amount of polypeptide is present in at least about 20%, at least about 30%, at least about 50%, at least about 80%, at least about 100%, at least about 200%, at least about 400%, at least about 600%, at least about 800%, or at least about 1000% greater than in the other sample.
  • prediction of gender refers to methods by which the skilled artisan can predict the condition in a subject. It does not refer to the ability to predict the gender with 100% accuracy. Instead the skilled artisan will understand that it refers to an high likelihood that the subject has the identified gender.
  • the prediction power might be less that 100%, for instance about 99.99%, about 99.9%, about 99.8%, about 99.7%, about 99.6%, about 99.5%, about 99%, about 98%, about 97%, about 96%, about 95%, about 90% or more, or about 80% or more.
  • the prediction power is at least 95%.
  • the term "about” in this context refers to +/-1%.
  • the present invention provides a method for predicting the gender of a mammalian subject, wherein said prediction is based on the expression profile of a transcript marker set.
  • the present invention further provides a kit for the prediction of the gender of a mammalian subject comprising means to assess the expression of said transcript marker set.
  • the present invention provides a comprehensive, robust and tissue independent gender- specific transcript marker set that can be applied for molecular gender determination.
  • the present invention provides for a method for predicting the gender of a mammalian subject, wherein said prediction is based on the expression profile of a group of genes.
  • Identifying or predicting the gender of a subject may be condisered as a diagnostic technique. Diagnostic methods differ in their sensitivity and specificity. The skilled artisan often makes a diagnosis, for example on the basis of one or more diagnostic indicators. In the present invention these are the expression profiles of the named genes. The presence, absence and amount of which indicate the gender of the subject.
  • the presenting invention in one aspect refers to a method, wherein the expression level of a group of genes with strong predictive power is determined within a subject to give the result for the subject .
  • the expression profile of the same genes is determined for a reference standard to give the reference result. The two results are compared.
  • the characteristic differences in expression level of the genes in the subject compared to the reference allow the prediction of the gender of the subject.
  • the method for predicting the gender of a mammalian subject is based on the expression profile of a group of genes comprising the ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), as well as the X-chromosome specific X (inactive)-specific transcript (XIST).
  • a group of genes comprising the ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D
  • the method for predicting the gender of a mammalian subject is based on the expression profile of the ribosomal protein S4, Y-linked 1 (RPS4Y1) and on the expression profile of the X-chromosome specific X (inactive)-specific transcript (XIST), and wherein said prediction is based on the ratio of the XIST expression to the RPS4Y1 expression.
  • the method for predicting the gender of a mammalian subject is based on the expression profile of a group of genes comprising the ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y- linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), the X- chromosome specific X (inactive)-specific transcript (XIST) and in addition the chromosome Y open reading frame 15A and 15B (CYorf15A and CYorf15B), the ubiquitin specific peptidase 9, Y-linked (fat facets-like, Drosophila) (USP9Y), the Zinc finger protein, Y-linked (ZFY), and the chromosome Y open reading frame 14 (CYorf14
  • the method for predicting the gender of a mammalian subject is based on the expression profile of a group of genes comprising the ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), the X-chromosome specific X (inactive)-specific transcript (XIST) and in addition the chromosome Y open reading frame 15A and 15B (CYorf15A and CYorf15B), the ubiquitin specific peptidase 9, Y-linked (fat facets-like, Drosophila) (USP9Y), the Zinc finger protein, Y-linked (ZFY), the chromosome Y open reading frame 14 (CYorf14),
  • Single or multiple determination of the gene expression of one or more of the named genes can be made either simultaneously or one after the other.
  • the expression of the genes is determined simultaneously.
  • the expression level of the genes is determined one after the other.
  • RPS4Y1 Y-linked 1
  • EIF1AY eukaryotic translation initiation
  • the present invention provides for a comprehensive, robust and tissue independent gender-specific transcript marker set.
  • the marker set consists of the ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y- linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARI D1 D), as well as the X-chromosome specific X (inactive)-specific transcript (XIST).
  • the marker set consists of the ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y- linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), the X-chromosome specific X (inactive)- specific transcript (XIST) and in addition of the chromosome Y open reading frame 15A and 15B (CYorfl ⁇ A and CYorf15B), the ubiquitin specific peptidase 9, Y-linked (fat facets-like, Drosophila) (USP9Y), the Zinc finger protein, Y-linked (ZFY), and the chromosome Y open reading frame 14 (CYorf14).
  • RPS4Y1 Y-linked 1
  • EIF1AY eukaryotic translation initiation factor 1A
  • the marker set of claim 10 consists of the ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y- linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), the X- chromosome specific X (inactive)-specific transcript (XIST) and in addition of the chromosome Y open reading frame 15A and 15B (CYorf15A and CYorf15B), the ubiquitin specific peptidase 9, Y-linked (fat facets-like, Drosophila) (USP9Y), the Zinc finger protein, Y-linked (ZFY), the chromosome Y open reading frame 14 (CYorf14), the ubiquitously transcribed tetratricopeptide repeat gene, Y
  • RPS4Y1 Y
  • the present invention provides for a threshold independent, ratio based gender-specific marker set.
  • the threshold independent, ratio based gender-specific marker set consists of the ribosomal protein S4, Y-linked 1 (RPS4Y1) and on the expression profile of the X-chromosome specific X (inactive)-specific transcript (XIST).
  • gene expression refers to the presence or amount of a specific gene including, but not limited to, mRNA, cDNA, or the polypeptide, peptide or protein expression product of a specific gene.
  • the gene expression is determined by measuring RNA levels. Gene expression may be detected using a PCR-based assay. Or in other aspects of the invention, reverse-transcriptase PCR (RT-PCR) is used to detect the expression of RNA.
  • RT-PCR reverse-transcriptase PCR
  • RNA is enzymatically converted to cDNA using a reverse-transcriptase enzyme.
  • the cDNA is then used as template for a PCR reaction.
  • PCR products can be detected by any suitable method including, but not limited to, gel electrophoresis and staining with a DNA-specific stain or hybridizaion to a labeled probe.
  • the quantitative RT-PCR with standardized mixtures of competitive templates can be utilized.
  • gene expression is detected using a hybridization assay.
  • a hybridization assay the presence or absence of marker is determined based on the ability of the nucleic acid from the sample to hybridize to a complementary nucleic acid molecule, e.g., an oligonucleotide probe.
  • a complementary nucleic acid molecule e.g., an oligonucleotide probe.
  • hybridization of a probe to the sequence of interest is detected directly by visualizing a bound probe, e.g., a Northern or Southern assay. In these assays, DNA (Southern) or RNA (Northern) is isolated.
  • the DNA or RNA is then cleaved with a series of restriction enzymes that cleave infrequently in the genome and not near any of the markers being assayed.
  • the DNA or RNA is then separated, e.g., on an agarose gel, and transferred to a membrane.
  • a labeled probe or probes e.g., by incorporating a radionucleotide, is allowed to contact the membrane under low-, medium- or high-stringency conditions. Unbound probe is removed and the presence of binding is detected by visualizing the labeled probe.
  • the gene expression is determined for one or more of the genes ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1D (JARID1 D), the X-chromosome specific X (inactive)-specific transcript (XIST), the chromosome Y open reading frame 15A and 15B (CYorf15A and CYorf15B), the ubiquitin specific peptidase 9, Y-linked (fat facets-like, Drosophila) (USP9Y), the Zinc finger protein, Y-linked (ZFY), the chromosome Y open reading frame 14 (CYorf14), the ubiquitously transcribed tetratricopeptide repeat gene, Y- linked (UTY),
  • the gene expression is determined by measuring polypeptide gene expression products.
  • gene expression is measured by identifying the amount of one or more polypeptides encoded by one of the genes ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), the X-chromosome specific X (inactive)-specific transcript (XIST), the chromosome Y open reading frame 15A and 15B (CYorf15A and CYorf15B), the ubiquitin specific peptidase 9, Y- linked (fat facets-like, Drosophila) (USP9Y), the Zinc finger protein, Y-linked (ZFY), the chromosome
  • the presence or amount of a protein or polypeptide or peptides is generally determined using specific antibodies and detecting specific binding.
  • Any suitable immunoassay may be utilized, for example, enzyme-linked immunoassays (ELISA), radioimmunoassays (RIAs), competitive binding assays, and the like.
  • ELISA enzyme-linked immunoassays
  • RIAs radioimmunoassays
  • Specific immunological binding of the antibody to the protein or polypeptide can be detected directly or indirectly.
  • Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody.
  • Indirect labels include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase and the like.
  • immobilized antibodies specific for the proteins or polypeptides are also contemplated by the present invention.
  • the antibodies can be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay place (such as microtiter wells), pieces of a solid substrate material (such as plastic, nylon, paper), and the like.
  • An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on solid support. This strip can then be dipped into the test sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.
  • the analysis of a plurality of genes and/or peptides of the present invention may be carried out separately or simultaneously with one test sample. In addition, one skilled in the art would recognize the value of testing multiple samples from the same individual.
  • a panel comprising the genes referenced above may be constructed to provide relevant information related to the identification of the gender.
  • Such a panel can be constructed preferably using the sequences of the genes ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (As p-G Iu-AIa-As p) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1D (JARID1 D), the X-chromosome specific X (inactive)-specific transcript (XIST), the chromosome Y open reading frame 15A and 15B (CYorf15A and CYorf15B), the ubiquitin specific peptidase 9, Y-linked (fat facets-like, Drosophila) (USP9Y), the Zinc finger protein, Y-linked (ZFY) 1 the chromosome Y open reading frame 14 (CY
  • Panels of genes according to the present invention may comprise one or more of the genes of the group consisting of the ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), as well as the X-chromosome specific X (inactive)-specific transcript (XIST) in any combination of single genes, pairs of any two of the genes, or groups of any three or four of the genes.
  • RPS4Y1 Y-linked 1
  • EIF1AY eukaryotic translation initiation factor 1A
  • EIF1AY eukaryotic translation initiation factor 1A box polypeptide 3
  • DDX3Y Y-linked box polypeptide 3
  • Jumonji AT rich interactive domain 1 D
  • Panels of genes according to the present invention may consist of one or more of the genes of the group consisting of the ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), as well as the X-chromosome specific X (inactive)-specific transcript (XIST) in any combination of single genes, pairs of any two of the genes, or groups of any three or four of the genes.
  • RPS4Y1 Y-linked 1
  • EIF1AY eukaryotic translation initiation factor 1A
  • EIF1AY eukaryotic translation initiation factor 1A box polypeptide 3
  • DDX3Y Y-linked box polypeptide 3
  • Jumonji AT rich interactive domain 1 D
  • the analysis of gene expression and/or determination of peptide levels can be carried out in a variety of physical formats as well.
  • the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples in a high throughput manner.
  • an array is provided to which probes that correspond in sequence to gene products, e.g., cDNAs, mRNAs, cRNAs, polypeptides and fragments thereof, can be specifically hybridized or bound at a known position.
  • gene products e.g., cDNAs, mRNAs, cRNAs, polypeptides and fragments thereof.
  • the array is a matrix in which each position represents a discrete binding site for a product encoded by a gene (e.g., a protein or RNA) preferably of the genes ribosomal protein S4, Y-linked 1 (RPS4Y1 ), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), the X-chromosome specific X (inactive)- specific transcript (XIST), the chromosome Y open reading frame 15A and 15B (CYorf15A and CYorf15B), the ubiquitin specific peptidase 9, Y-linked (fat facets-like, Drosophila) (USP9Y), the Zinc finger protein, Y-linked (ZFY), the chromosome Y open
  • the "binding site”, hereinafter “site” is a nucleic acid or nucleic acid analogue to which a particular cognate cDNA can specifically hybridize.
  • the nucleic acid or analogue of the binding site can be, e.g., a synthetic oligomer, a full-length cDNA, a less than full-length cDNA or a gene fragment.
  • the present invention provides a kit for the analysis of gene expression and/or peptide levels. Such a kit preferably comprises devices and reagents for the analysis of at least one test sample and instructions for performing the assay.
  • kits may contain one or more means for converting gene expression and/or amounts of peptides to a diagnosis or prognosis of coronary artery disease in a subject. Comparison of the subject's gene expression pattern, with the controls or reference standards, would indicate whether the subject is male or female.
  • the present invention provides for a kit for the prediction of the gender of a mammalian subject.
  • kits comprise devices and reagents for measuring gene expression and/or determining peptide levels in a subjects sample and instructions for performing the assay and interpreting the results.
  • Such kits preferably contain sufficient reagents to perform on or more such determinations.
  • the kit comprises means to assess the expression of the ribosomal protein S4, Y-linked 1 (RPS4Y1 ) gene and of the X- chromosome specific X (inactive)-specific transcript (XIST).
  • the kit comprises means to assess the expression of ribosomal protein S4, Y-linked 1 (RPS4Y1 ), the eukaryotic translation initiation factor 1A 1 Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y- linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), as well as the X-chromosome specific X (inactive)-specific transcript (XIST).
  • RPS4Y1 Y-linked 1
  • EIF1AY eukaryotic translation initiation factor 1A 1 Y-linked box polypeptide 3
  • DDX3Y Y- linked box polypeptide 3
  • Jumonji AT rich interactive domain 1 D
  • JARID1 D the X-chromosome specific X (inactive)-specific transcript
  • the kit comprises means to assess the expression of ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), as well as the X-chromosome specific X (inactive)-specific transcript (XIST) and in addition the chromosome Y open reading frame 15A and 15B (CYorfl ⁇ A and CYorfl ⁇ B), the ubiquitin specific peptidase 9, Y-linked (fat facets-like, Drosophila) (USP9Y), the Zinc finger protein, Y-linked (ZFY), and the chromosome Y open reading frame 14 (CYorf14).
  • RPS4Y1 Y-linked 1
  • EIF1AY eukaryotic translation
  • the kit comprises means to assess the expression of ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (As p-G Iu -Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), as well as the X-chromosome specific X (inactive)-specific transcript (XIST) and in addition the chromosome Y open reading frame 15A and 15B (CYorf15A and CYorf15B), the ubiquitin specific peptidase 9, Y-iinked (fat facets-like, Drosophila) (USP9Y), the Zinc finger protein, Y-linked (ZFY), the chromosome Y open reading frame 14 (CYorf14), ubiquitously transcribed tetratricopeptid
  • kits contain antibodies specific for at least one gene, preferably from ribosomal protein S4, Y-linked 1 (RPS4Y1 ), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y- linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1 D), the X- chromosome specific X (inactive)-specific transcript (XIST), the chromosome Y open reading frame 15A and 15B (CYorfi ⁇ A and CYorfi ⁇ B), the ubiquitin specific peptidase 9, Y-linked (fat facets-like, Drosophila) (USP9Y), the Zinc finger protein, Y-linked (ZFY), the chromosome Y open reading frame 14 (CYorf14), the ubiquitously transcribed tetratricopeptide repeat gene,
  • RPS4Y1
  • kits contain reagents specific for the detection of nucleic acid, e.g., oligonucleotide probes or primers.
  • the kits contain all of the components necessary to perform a detection assay, including all controls and instructions for performing assays and for analysis of results.
  • the kits contain instructions including a statement of intended use as required by the Environmental Protection Agency or U.S. Food and Drug Administration (FDA) for the labeling of in vitro diagnostic assays and/or of pharmaceutical or food products.
  • FDA Food and Drug Administration
  • an object of the present invention is to provide a comprehensive, robust and tissue independent gender-specific transcript marker set that can be applied for molecular gender determination in human peripheral blood but also in other healthy and diseased tissues. It was expected that many of the discovered gender specific transcripts would potentially be encoded by autosomes and/or represent genes whose expression may be dependent on the physiological status of the organism driven by exogenous factors such as food, lifestyle, medication, or endogenous factors among them age or hormone levels. Since such transcripts were not expected to represent the most robust and tissue independent gender markers, the present inventors intended to focus the analysis primarily on heterosome encoded, constitutively expressed, transcripts.
  • NHPs non-human primates
  • This prediction can be performed using different algorithms and/or statistical methods well known in the art.
  • methods and/or algorithms are parametric models such as logistic regression, linear/quadratic discriminant analysis, nonparametric models, e.g. k- nearest neighbors, nearest centroid, machine learning approaches such as support vector maschines, neural networks, or simple heuristic rules (Hastie et al. 2003).
  • An expression profile of a group of genes at the level of their transcripts or proteins can be assessed by methods well known in the art. Examples are approaches that were primarily developed and applied to analyze individual analytes but at the same time may potentially be multiplexed such Northern blot hybridization or (quantitative) polymerase chain reaction, in situ-hybridization for transcripts, or western blot hybridization, immune-histochemistry, or enzymatic activity assays, for proteins (e.g. Sambrook et al. 2000, Simard et al. 2007, lnfanger et al 2007).
  • the mammalian subject can be any mammalian, it can for instance be a human, a dog, a mouse, a rat, a hamster, a rabbit, a cow, a horse, a pig, a camel, or a monkey.
  • the mammalian subject is human or non-human primate.
  • the subject is human.
  • Means to assess the expression of the ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y), and the Jumonji, AT rich interactive domain 1 D (JARID1D), as well as the X-chromosome specific X (inactive)-specific transcript (XIST) extended panel are well known in the art.
  • Said means comprise, for instance, nucleic acid probes and primers. Expression could also be measured at the protein level using, e.g. antibodies, for instance by ELISA, or specific metabolites that may have derived from potential catalytic activities of .
  • a comprehensive, robust and tissue independent gender-specific transcript marker set is a set of messenger RNA transcript that allows a accurate prediction of the gender of a subject.
  • DDX3Y DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked; synonym
  • EIF1AX Eukaryotic translation initiation factor 1A, X-linked
  • EIF1AY Eukaryotic translation initiation factor 1A 1 Y-linked
  • OGT O-linked N-acetylglucosamine (GIcNAc) transferase (UDP-N- acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase)
  • RNA samples were preserved by snap-freezing or treatment with RNA later according to the manufacturers instructions (http://server1.ambion.com/techlib/resources/RNAIater/) immediately after excision. Cancerous samples were defined to contain a majority of cancerous cells. Samples were stored at approximately -80 0 C until further analysis.
  • the sequence clusters were created from the UniGene database (Build 133, April 20, 2001 ) and then refined by analysis and by comparison with a number of other publicly available databases, including the Washington University Expresses Sequence Tag (EST trace repository and the University of California, Santa Cruz Golden-Path human genome database (April 2001 release).
  • the DNA microarrays were processed as recommended by the manufacturer (GeneChip® Expression Analysis Technical Manual) using the recommended cRNA labeling, GeneChip® hybridization, wash, and stain protocols (Affymetrix, Inc.).
  • the GeneChip arrays were scanned using a GS 3000 scanner (Affymetrix, Inc.). Quality control of images was performed by visual inspection and expert judgment.
  • the primary raw data images, the .dat- files, were processed to .eel-files and to numerical signal values (.chip files) using the DNA Microarray Analysis Suite 5 (MAS5) software package (Affymetrix, Inc.).
  • the training data set was derived from MAS5 normalized numerical signal values obtained from male and female human blood samples and analyzed using summary statistics (per gender), exploratory graphical displays. Analysis of Variance (ANOVA) was based on Iog2- transformed expression intensities.
  • the individual markers selected for gender prediction were identified based on the size of the following ratio median (expression intensities of males) / median (expression intensities of females) or the invers ratio; with successive selection criteria including a median MAS5 expression intensity larger then twice the background level in at least one gender group, a p-value ⁇ 0.001 in the ANOVA, as well as small intra-group variances (i.e. no or only very small overlap between the intensity distributions of the two gender groups per individual marker). No additional filter criteria were applied (Quackenbush 2002, Wit and McClure 2004).
  • Probe set annotations including functional annotations was obtained from the NetAffx data base (Liu et al. 2003; https://www.affymetrix.com/analvsis/netaffx/index.affx; release for the human genome U133 plus 2.0 array download as of the 17 th July, 2007 GenBank 15-Feb-07, 158; RefSeq 05-Mar-07 22; UniProt 12-Jun-07 11.1 , Entrez Gene 5-Jun-07; Ensembl Apr- 07 44; UniGene 01-Mar-07 201 Homo sapiens).
  • RT-PCR Real time-PCR
  • Primers were designed using the Primer Express software version 2.0 (Applied Biosystems Applied Biosystems, Foster City, U. S; table 1.) and synthesized by Microsynth AG (Balgach, Switzerland). The specificity of the selected primers was confirmed using the NCBI Nucleotide BLAST web-tool (National Center for Biotechnology Information, Bethesda, MD, U.S.A., http://www.ncbi. nlm.nih.qov/BLAST ⁇ . Approximately 1 ⁇ g total RNA from each sample was converted into cDNA using the High-Capacity cDNA reverse transcription kit with RNAse inhibitor (Applied Biosystems).
  • ID identifier
  • Gene Name or Symbol Gene name or symbol according to the HUGO Gene Nomenclature Committee database (HGNC, Eyre et al. 2006).
  • the predictive power of selected transcript markers was assessed using blinded test data sets.
  • DNA microarray data we used two approaches. First, based on MAS5 normalized expression signals and intensity-threshold values were obtained for each individual marker. Predictions were made for each individual marker as well as a marker panel, based on the majority vote concept (Penrose 1946; May 1952; MacKay 2002; Karchin et al. 2007).
  • the nine most prominently expressed members of this gene list are the ribosomal protein S4, Y-linked 1 (RPS4Y1), the eukaryotic translation initiation factor 1A, Y-linked (EIF1AY), the DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y- linked (DDX3Y), the Jumonji, AT rich interactive domain 1 D (JARID1 D), the chromosome Y open reading frame 15A and 15B (CYorf15A and CYorf15B), the ubiquitin specific peptidase 9, Y-linked (fat facets-like, Drosophila) (USP9Y), the Zinc finger protein, Y-linked (ZFY) 1 and the chromosome Y open reading frame 14 (CYorf14) (172.4 to 6.1 -fold higher in males).
  • RPS4Y1 Y-linked 1
  • EIF1AY eukaryotic translation initiation factor 1A
  • EIF1AY e
  • Y-chromosome located genes were only marginally higher expressed in males (3.6 to 2-fold), the ubiquitously transcribed tetratricopeptide repeat gene, Y-linked (UTY), the protein kinase, Y-linked (PRKY), the BCL6 co-repressor-like 2 (BCORL2), and the testis- specific transcript, Y-linked 15 (TTTY15).
  • UTY ubiquitously transcribed tetratricopeptide repeat gene
  • PRKY protein kinase
  • BCL6 co-repressor-like 2 BCORL2
  • TTTY15 testis- specific transcript
  • genes were represented by up to three redundant probe sets, EIF1AY, DDX3Y, CYorf15A, CYorf15B, USP9Y, ZFY, and UTY, all were up-regulated with slightly different fold-changes most likely related to the exact probe sequences and position of the probes with regard to the transcripts.
  • the seven other X-linked genes were marginally higher in females (3.5 to 2-fold), Moesin (MSN), X (inactive)-specific transcript, antisense (TSIX), the Fanconi anemia, complementation group (FANCB), the brain expressed, X-linked 1 (BEX1), the vestigial like 1 (Drosophila) (VGLL1), the transcription elongation factor A (Sll)-like 7 (TCEAL7), and the O-linked N-acetylglucosamine (GIcNAc) transferase (UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase) (OGT).
  • GCT O-linked N-acetylglucosamine
  • Figure 1 revealed either no or only a very minor overlap between the data distribution between both genders. Only in case of DDX3Y (probe set 205000_at, figure 1c.) there was no complete segregation into the proper sexes. Two of the 65 male donors (3.1%) exhibited no clear separation of between genders, which was not thought to lead to a major disturbance of the predictive power of this marker and the overall approach.
  • the REDJX marker panel based approach was used.
  • a threshold independent, ratio based algorithm was formed based on the ratio of the X- chromosome located marker transcript, XIST represented by probe set 221728_x_at, and the RPS4Y1 transcript represented by probe set 224588_at. For a reliable prediction the ratios were defined to be > 2 for females or ⁇ 0.5 for males.
  • a second set of samples was derived from 55 human donors (24 males, 31 females, test set 3). From each donor two samples were analyzed, one from a cancerous tissue sample and one from healthy tissue from the same organ of the same donor. In total 1 10 samples from 11 further human tissues was analyzed, breast (20 samples), colon (4), large intestine (12), larynx (12), lip (2), liver (16), lung (20), ovary (16), parotid gland (2), pharynx (4), and rectum (2) (table 7).
  • Predictive power was 100 % in all healthy samples for all individual markers as well as the marker panel and the XIST 224588_at/ RPS4Y1 201909_at ratio for all healthy samples, as well for the REDJX marker panel and the XIST 224588_at/ RPS4Y1 201909_at ratio.
  • predictive power of 100% was achieved using the REDJX marker panel and the XIST 224588_at/ RPS4Y1 201909_at ratio using the established thresholds (table 7).
  • none of the individual markers was fully predictive in all diseased samples. In total there were 32 mispredicted cancerious samples representing 6.5% of all 495 (55 x 9) individual marker based predictions. Per individual marker the number of erroneous predictions varied between 1 and 7, representing between 1 8% and 12.7% of all 55 donors (figure 2).
  • RT-PCR experiments for two selected gender specific marker genes were conducted.
  • Several algorithms were empirically developed (table 8) based on the values observed in the present experiment.
  • XIST probeset 227671_at fully failed.
  • the XIST (224588_at)/ RPS4Y1 (201909_at) ratio based algorithm gave partially erroneous results and therefore could not properly predict the genders.
  • GeneChip genome wide DNA microarray
  • the present inventors identified a total of 242 probe sets, mapping to 201 genes as annotated in NetAffx (Liu et al., 2003) from comparing female and male samples.
  • the present inventors focused on heterosome encoded, constitutively expressed transcripts.
  • the list of gender specific expressed genes including the Y- and X- linked markers (table 11) differ profoundly from those published elsewhere (Galfalvy et al. 2003, Tang et al. 2004, Vawter et al. 2004; Ostrowski et al. 2006, Kim et al. 2006, lsensee at al. 2007).
  • the present inventors found 1 18 probe sets mapping to 88 HGNC annotated genes that were higher expressed compared to females, 23 of these probe sets pointed to 13 Y- chromosome located genes.
  • the nine most prominently expressed Y-chromosome located genes, RPS4Y1 , EIF1AY, DDX3Y, JARID1 D, CYorf15A, CYorf15B, USP9Y, ZFY, and CYorf14 al! belong to the class of X-degenerate genes of the Y-chromosome encoded transcripts and are described to be ubiquitously expressed (Skaletzky et al, 2003). This further corroborates their potential as ubiquitous, tissue-independent molecular markers.
  • TTTY15 belongs to the class of ampliconic genes that are known to be testes specific (Skaletzky et al, 2003). The fact that the present inventors found TTTY15 expressed in the blood suggests a less specific expression than previously thought and/or potentially another function outside of the testis.
  • the present inventors focused primarily on heterosome encoded, constitutively expressed transcripts to avoid transcripts whose expression may be determined by ever changing endogenous or exogenous factors. The results obtained confirmed the appropriateness of the selected approach to address the questions asked. Whether or not the autosome-related transcripts would also represent useful markers remains subject of further research. From the unique list of marker transcripts discovered from microarray data, the present inventors selected a subset of 9 probe sets mapping to 5 differentially expressed transcripts for gender prediction, the Y-chromosome specific RPS4Y1 , EIF1AY, DDX3Y, and JARID1 D (highly expressed in males), and the X- chromosome specific XIST (notably higher expressed in females) for further investigations.
  • NHPs non-human primates
  • Biomarker qualification supporting drug development is an emerging scientific and regulatory topic that was initially triggered by the new guideline of the US Food and Drug Administration (FDA) for pharmacogenomic data submissions (US FDA, 2006a.
  • FDA US Food and Drug Administration
  • US FDA, 2006a An overview of valid genomic biomarkers in the context of approved drug labels is accessible through the regulators web site (US FDA, 2006b).
  • How the qualification of biomarkers should look like is a matter of ongoing discussions (Goodsaid and Frueh, 2006, 2007, Williams et al 2006, Wagner et al. 2007, Marrer and Dieterle, 2007). What is obvious from the current debate is that biomarker qualification has to be seen in the context of the planned usage.
  • Tonin PN Global analysis of chromosome X gene expression in primary cultures of normal ovarian surface epithelial cells and epithelial ovarian cancer cell lines, lnt J Oncol. 2007
  • Bianchi DW Wataganara T, Lapaire O, Tjoa ML, Maron JL, Larrabee PB, Johnson KL. Fetal nucleic acids in maternal body fluids: an update. Ann N Y Acad Sci. 2006 Sep; 1075:63-73. Brown, C. J. et al. A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome. Nature 349, 38-44 (1991).
  • X and Y chromosomes escape from X inactivation and possible implications for Turner syndrome. Cell 63: 1205-1218, 1990.
  • RNA maps reveal new RNA classes and a possible function for pervasive transcription. Science. 2007 Jun 8;316(5830):1484-8. Epub 2007 May 17. lnfanger M, Ulbrich C, Baatout S, Wehland M, Kreutz R, Bauer J, Grosse J, Vadrucci S 1 Cogoli A, Derradji H, Neefs M, Kusters S, Spain M, Paul M, Grimm D. Modeled gravitational unloading induced downregulation of endothelin-1 in human endothelial cells. J Cell Biochem. 2007 Aug 15; 101 (6): 1439-55.
  • Liang P, Meade JD, Pardee AB A protocol for differential display of mRNA expression using either fluorescent or radioactive labeling. Nat Protoc. 2007;2(3):457-70.
  • Liu J, Lissens W, Devroey P, Van Steirteghem A, Liebaers I Amplification of X- and Y- chromosome-specific regions from single human blastomeres by polymerase chain reaction for sexing of preimplantation embryos. Hum Reprod. 1994 Apr;9(4):716-20.
  • Kalaitzopoulos D Komura D, MacDonald JR, Marshall CR, Mei R, Montgomery L, Nishimura
  • the male-specific region of the human Y chromosome is a mosaic of discrete sequence classes.
  • Simard LR Belanger MC, Morissette S, Wride M, Prior TW, Swoboda KJ. Preclinical validation of a multiplex real-time assay to quantify SMN mRNA in patients with SMA.
  • Tsui NB Dennis Lo YM. Placental RNA in maternal plasma: toward noninvasive fetal gene expression profiling. Ann N Y Acad Sci. 2006 Sep;1075:96-102. Review.
  • Venter JC Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, Yandell M,
  • Wataganara T LeShane ES, Chen AY, Borgatta L, Peter I, Johnson KL, Bianchi DW.
  • Plasma gamma-globin gene expression suggests that fetal hematopoietic cells contribute to the pool of circulating cell-free fetal nucleic acids during pregnancy.
  • Circulating cell-free fetal nucleic acid analysis may be a novel marker of fetomaternal hemorrhage after elective first-trimester termination of pregnancy.
  • Circulating placental RNA in maternal plasma is associated with a preponderance of 5' mRNA fragments: implications for noninvasive prenatal diagnosis and monitoring. Clin Chem. 2005

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Abstract

La présente invention concerne un procédé pour prédire le genre d'un sujet mammifère, la prédiction étant basée sur le profil d'expression de marqueurs de transcrits.
PCT/EP2008/062529 2007-09-20 2008-09-19 Marqueurs de transcrits spécifiques du genre, robustes et indépendants des tissus, pour la détermination moléculaire du genre WO2009037337A1 (fr)

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