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WO2025078620A1 - Évaluation d'un trouble du spectre autistique par analyse de polyadénylation alternative - Google Patents

Évaluation d'un trouble du spectre autistique par analyse de polyadénylation alternative Download PDF

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Publication number
WO2025078620A1
WO2025078620A1 PCT/EP2024/078720 EP2024078720W WO2025078620A1 WO 2025078620 A1 WO2025078620 A1 WO 2025078620A1 EP 2024078720 W EP2024078720 W EP 2024078720W WO 2025078620 A1 WO2025078620 A1 WO 2025078620A1
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Prior art keywords
asd
apa
parameter
utr
sample
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Inventor
Ana Martin-Villalba
Simon Anders
Andre LOPES MARTINS MACEDO
Nikhil GEORGE OOMMEN
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Deutsches Krebsforschungszentrum DKFZ
Universitaet Heidelberg
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Deutsches Krebsforschungszentrum DKFZ
Universitaet Heidelberg
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method for assessing an autism spectrum disorder (ASD) in a sample from a subject, the method comprising (a) determining a parameter of alternative polyadenylation (APA parameter) in said sample; (b) comparing the APA parameter determined in step (a) to a reference, and (c) based on the comparison in step (b), assessing said ASD.
  • the present invention also relates to polynucleotides, means for determining an APA parameter for use in diagnosis, databases, devices, and further methods related thereto.
  • a minority of ASD cases are syndromic forms caused by highly penetrant single-gene mutations or chromosomal abnormalities; these forms are often accompanied by additional phenotypes, such as intellectual disability, epilepsy and craniofacial dysmorphology.
  • most ASD cases are idiopathic, with genetic causality residing in polygenic risk involving small effect-size variants in hundreds of genes.
  • APA alternative polyadenylation
  • the present invention relates to a method for assessing an autism spectrum disorder (ASD) in a sample from a subject, the method comprising (a) determining a parameter of alternative polyadenylation (APA parameter) in said sample; (b) comparing the APA parameter determined in step (a) to a reference, and (c) based on the comparison in step (b), assessing said ASD.
  • ASD autism spectrum disorder
  • the present invention relates to a method for assessing a light form of an autism spectrum disorder (ASD), preferably ASD level 1, in a sample from a subject, the method comprising (a) determining a parameter of alternative polyadenylation (APA parameter) in said sample; (b) comparing the APA parameter determined in step (a) to a reference, and (c) based on the comparison in step (b), assessing said light form of ASD, wherein said APA parameter preferably comprises at least one mRNA 3'UTR length of ATP5F1E, BUB3, CDK10, CENPB, COX7B, ELOB, FAM3A, IFIT1, JUND, NSMCE1, OSTF1, PDCD10, PRR13, QSOX1, RPL32, RPL35A, RPL36, RPLP1, RPN1, RPS15A, SERTAD3, SLC25A44, TSC22D3, UBE2A, ZBTB7B, EEF1A1, EIF4
  • ASSD autism
  • the term "multitude”, as referred to herein, relates to a number of more than one, i.e. at least two, preferably at least three, more preferably at least four, even more preferably at least five. A multitude may however, also be a number of at least ten, at least 25, at least 100, or more.
  • the terms "preferably”, “more preferably”, “most preferably”, “particularly”, “more particularly”, “specifically”, “more specifically” or similar terms are used in conjunction with optional features, without restricting further possibilities. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using alternative features.
  • features introduced by "preferably” or similar expressions are intended to be optional features, without any restriction regarding further embodiments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such way with other optional or non-optional features of the invention.
  • the methods specified herein below, preferably, are in vitro methods. The method steps may, in principle, be performed in any arbitrary sequence deemed suitable by the skilled person, but preferably are performed in the indicated sequence; also, one or more, preferably all, of said steps may be assisted or performed by automated equipment. Moreover, the methods may comprise steps in addition to those explicitly mentioned above.
  • the term "about” relates to the indicated value with the commonly accepted technical precision in the relevant field, preferably relates to the indicated value ⁇ 20%, more preferably ⁇ 10%, most preferably ⁇ 5%.
  • the term “essentially” indicates that deviations having influence on the indicated result or use are absent, i.e. potential deviations do not cause the indicated result to deviate by more than ⁇ 20%, more preferably ⁇ 10%, most preferably ⁇ 5%.
  • “consisting essentially of” means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention.
  • compositions defined using the phrase “consisting essentially of” encompasses any known acceptable additive, excipient, diluent, carrier, and the like.
  • a composition consisting essentially of a set of components will comprise less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, most preferably less than 0.1% by weight of non-specified component(s).
  • the degree of identity e.g. expressed as "%identity" between two biological sequences, preferably DNA, RNA, or amino acid sequences, can be determined by algorithms well known in the art.
  • the degree of identity is determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the sequence it is compared to for optimal alignment.
  • the percentage is calculated by determining, preferably over the whole length of the polynucleotide or polypeptide, the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970), by the search for similarity method of Pearson and Lipman (1988), by computerized implementations of these algorithms (e.g. BLAST, GAP, BESTFIT, PASTA, or TFASTA), or by visual inspection.
  • GAP and BESTFIT are preferably employed to determine their optimal alignment and, thus, the degree of identity.
  • the default values of 5.00 for gap weight and 0.30 for gap weight length are used.
  • the Basic Local Alignment Search Tool (BLAST) implementation is used with default parameter values for alignment.
  • the term "essentially identical” indicates a %identity value of at least 80%, preferably at least 90%, more preferably at least 98%, most preferably at least 99%. As will be understood, the term essentially identical includes 100% identity. The aforesaid applies to the term “essentially complementary” mutatis mutandis.
  • fragment of a biological macromolecule, preferably of a polynucleotide or polypeptide, is used herein in a wide sense relating to any sub-part, preferably subdomain, of the respective biological macromolecule comprising the indicated sequence, structure and/or function.
  • the term includes sub-parts generated by actual fragmentation of a biological macromolecule, but also sub-parts derived from the respective biological macromolecule in an abstract manner, e.g. in silico.
  • an Fc or Fab fragment but also e.g. a single-chain antibody, a bispecific antibody, and a nanobody may be referred to as fragments of an immunoglobulin.
  • the compounds specified, in particular the polynucleotides and polypeptides may be comprised in larger structures, e.g. may be covalently or non-covalently linked to further sequences, carrier molecules, retardants, and other excipients.
  • polypeptide refers to a molecule consisting of several, typically at least 20 amino acids that are covalently linked to each other by peptide bonds. Molecules consisting of less than 20 amino acids covalently linked by peptide bonds are usually considered to be "peptides".
  • the polypeptide comprises of from 50 to 1000, more preferably of from 75 to 1000, still more preferably of from 100 to 500, most preferably of from 110 to 400 amino acids.
  • the polypeptide is comprised in a fusion polypeptide and/or a polypeptide complex.
  • polynucleotide is known to the skilled person.
  • the term includes nucleic acid molecules comprising or consisting of a nucleic acid sequence or nucleic acid sequences as specified herein, optionally further comprising a chemical molecule allowing detection of the polynucleotide, i.e. an indicator agent, such as a dye.
  • the polynucleotide of the present invention shall be provided, preferably, either as an isolated polynucleotide, i.e. isolated from its natural context, or in genetically modified form.
  • the polynucleotide preferably, is DNA, including cDNA, or is RNA.
  • the term encompasses single as well as double stranded polynucleotides, as well as circularly closed and linear polynucleotides.
  • the polynucleotide is a chimeric molecule, i.e., preferably, comprises at least one nucleic acid sequence, preferably of at least 20 bp, more preferably at least 100 bp, heterologous to the residual nucleic acid sequence(s) or being an artificial nucleic acid sequence.
  • comprised are also chemically modified polynucleotides including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides or artificial modified ones such as biotinylated polynucleotides, peptide nucleic acids, locked nucleic acids, and the like.
  • the polynucleotide may, in principle, have any length deemed appropriate by the skilled person and/or as specified herein.
  • a polynucleotide comprising less than 25 nucleotides may also be referred to as an "oligonucleotide”
  • a probe polynucleotide with a length of less than 25 nucleotides may also be referred to as a "probe oligonucleotide”
  • a primer polynucleotide with a length of less than 25 nucleotides may be referred to as a "primer oligonucleotide” or as a "primer”.
  • polynucleotide includes polynucleotide variants.
  • polynucleotide variant relates to a variant of a polynucleotide referred to herein comprising a nucleic acid sequence characterized in that the sequence can be derived from the aforementioned specific nucleic acid sequence by at least one nucleotide substitution, addition and/or deletion, wherein the polynucleotide variant shall have the activity as specified for the specific polynucleotide.
  • said polynucleotide variant is an ortholog, a paralog or another homolog of the specific polynucleotide.
  • polynucleotide variant is or is derived from a non-naturally occurring allele of the specific polynucleotide.
  • Polynucleotide variants also encompass polynucleotides comprising a nucleic acid sequence which is capable of hybridizing to the aforementioned specific polynucleotides, preferably, under stringent hybridization conditions. These stringent conditions are known to the skilled worker and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6.
  • variants include polynucleotides comprising nucleic acid sequences which are at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, most preferably at least 99%, identical to the specifically indicated nucleic acid sequences.
  • the percent identity values are, preferably, calculated over the entire nucleic acid sequence region, preferably as specified herein above.
  • the polynucleotides of the present invention either consist, essentially consist of, or comprise the aforementioned nucleic acid sequences. Thus, they may contain further nucleic acid sequences as well.
  • the polynucleotides and variants thereof may be comprised in a vector.
  • the method for assessing of the present invention preferably, is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to providing a sample for step (a), or performing additional diagnostic steps before or after step (c).
  • ASD autism spectrum disorder
  • ASD is known to the skilled person to relate to a diverse group of conditions of human subjects characterized by at least some degree of difficulty with social interaction and communication. Other characteristics are atypical patterns of activities and behaviors, such as difficulty with transition from one activity to another, a focus on details and unusual reactions to sensations.
  • ASD is autism, Asperger's syndrome, or childhood disintegrative disorder.
  • ASD is a light form of ASD, wherein the term "light form of ASD" preferably relates to a form of ASD referred to as ASD level 1.
  • ASD may be ASD level 1.
  • the aforesaid assessment is provided at a young age of the subject, preferably before the age of 10 years, more preferably before the age of five years, still more preferably before the age of 3 years, even more preferably before the age of two years, most preferably before the age of one year. Since the assessment may also be performed e.g. on a blood sample, it may in a preferred embodiment also be performed shortly after birth, e.g. within two weeks, more preferably within four weeks, still more preferably within three months, even more preferably within six months, most preferably within twelve months after birth. In a further preferred embodiment, the assessment is performed before the subject develops speech, i.e. preferably before the age of one year.
  • tissue or organ samples may be obtained from any tissue or organ by, e.g., biopsy, surgery, or any other method deemed appropriate by the skilled person.
  • Separated cells may be obtained from the body fluids, such as lymph, blood, cerebrospinal fluid (CSF) and other, or from the tissues or organs by separating techniques such as centrifugation or cell sorting.
  • the sample is a non-brain sample, i.e. is not a sample of intracranial tissue; thus, the sample preferably does not comprise neurons and/or glial cells.
  • subject as referred to herein, relates to a mammal, preferably a human.
  • an APA parameter may be the average length of the 3' untranslated region (3'UTR) of transcripts of a marker gene, preferably selected from the marker genes of Table 1.
  • a parameter of APA may also be determined by determining the presence or absence of nucleic acid sequences which are present in a normal (or extended) variant of a 3'UTR, but not in a shortened (or normal) variant of said 3'UTR. Also in such case, determinations of 3'UTRs from more than one marker gene may be combined into a score.
  • the APA parameter comprises at least one mRNA 3'UTR length of RPN1, UBE2A and/or MKRN2.
  • the APA parameter comprises mRNA 3'UTR lengths of RPN1, UBE2A, and MKRN2.
  • the APA parameter comprises mRNA 3'UTR lengths of RPN1, UBE2A, MKRN2, UHMK1, IER3, BUB3, MLX, CA2, ITM2A, and/or NT5C3A. Still more preferably, the APA parameter comprises mRNA 3'UTR lengths of at least two, preferably at least three, more preferably at least four, even more preferably at least five, still more preferably at least six, still more preferably at least seven, still more preferably at least eight, still more preferably at least nine, most preferably all ten genes selected from the list consisting of RPN1, UBE2A, MKRN2, UHMK1, IER3, BUB3, MLX, CA2, ITM2A, and NT5C3A.
  • the APA parameter comprises mRNA 3'UTR lengths of the genes RPN1, UBE2A, MKRN2, UHMK1, IER3, BUB3, MLX, CA2, ITM2A, and NT5C3A.
  • the APA parameter comprises at least one mRNA 3'UTR length of ATP5F1E, BUB3, CDK10, CENPB, COX7B, ELOB, FAM3A, IFIT1, JUND, NSMCE1, OSTF1, PDCD10, PRR13, QSOX1, RPL32, RPL35A, RPL36, RPLP1, RPN1, RPS15A, SERTAD3, SLC25A44, TSC22D3, UBE2A, and/or ZBTB7B or of ATP5F1E, BUB3, CDK10, CENPB, COX7B, ELOB, FAM3A, IFIT1, JUND, NSMCE, and/or ZBTB7B or of ATP5F1E, BUB3, CD
  • the APA parameter comprises mRNA 3'UTR lengths of at least five, preferably at least ten, more preferably at least 15, even more preferably at least 20, still more preferably at least 25, still more preferably at least all genes selected from the list consisting of ATP5F1E, BUB3, CDK10, CENPB, COX7B, ELOB, FAM3A, IFIT1, JUND, NSMCE1, OSTF1, PDCD10, PRR13, QSOX1, RPL32, RPL35A, RPL36, RPLP1, RPN1, RPS15A, SERTAD3, SLC25A44, TSC22D3, UBE2A, and ZBTB7B; or consisting of ATP5F1E, BUB3, CDK10, CENPB, COX7B, ELOB, FAM3A, IFIT1, JUND, NSMCE1, OSTF1, PDCD10, PRR13, QSOX
  • the APA parameter comprises mRNA 3'UTR lengths of ATP5F1E, BUB3, CDK10, CENPB, COX7B, ELOB, FAM3A, IFIT1, JUND, NSMCE1, OSTF1, PDCD10, PRR13, QSOX1, RPL32, RPL35A, RPL36, RPLP1, RPN1, RPS15A, SERTAD3, SLC25A44, TSC22D3, UBE2A, and ZBTB7B or of ATP5F1E, BUB3, CDK10, CENPB, COX7B, ELOB, FAM3A, IFIT1, JUND, NSMCE1, OSTF1, PDCD10, PRR13, QSOX1, RPL32, RPL35A, RPL36, RPLP1, RPN1, RPS15A, SERTAD3, SLC25A44, TSC22D3, UBE2A, ZBTB
  • marker gene relates to a gene, preferably of a subject and preferably expressed by cells in a sample, from which at least one transcript is transcribed which shows differential polyadenylation in subjects suffering from ASD as compared to subjects not suffering from ASD.
  • marker genes preferably, are marker genes of ASD and, more preferably, determining an APA parameter of a gene product thereof allows the assessment to be made. Methods for identifying a marker gene as referred to herein are provided on an exemplary basis herein in the Examples.
  • Preferred marker genes are provided in Table 1.
  • marker genes are provided in Table 14; thus, in a further prefered embodiment, the marker gene(s) is/are selected independently from Table 1 and/or Table 14.
  • the marker genes of Table 1 and Table 14 are, in principle known to the skilled person and can be accessed by the indicated accession numbers. Unless indicated otherwise, all database accession numbers in this description refer to the respective entry current at the filing date of this description. It is understood by the skilled person that the marker genes are referenced as biomarkers, not as specific polynucleotides or polypeptides. Accordingly, the gene products of the marker genes, in particular polynucleotides and polypeptides having the specific sequences deposited under the database accession numbers, are to be understood as exemplary sequences representing a marker gene.
  • variant polynucleotides which vary due to at least one nucleotide addition, substitution and/or deletion from the polynucleotide having the specific sequence deposited in the database as long as they are also suitable as biomarkers as discussed above.
  • the variant polynucleotides are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the specific polynucleotides.
  • the APA parameter is determined based on all RNA transcript gene products of a marker gene present in a sample.
  • a multitude of marker genes is used in the assessment.
  • the marker gene is RPN1, UBE2A and/or MKRN2, more preferably is RPN1, UBE2A, MKRN2, UHMK1, IER3, BUB3, MLX, CA2, ITM2A, and/or NT5C3A.
  • the marker gene is ATP5F1E, BUB3, CDK10, CENPB, COX7B, ELOB, FAM3A, IFIT1, JUND, NSMCE1, OSTF1, PDCD10, PRR13, QSOX1, RPL32, RPL35A, RPL36, RPLP1, RPN1, RPS15A, SERTAD3, SLC25A44, TSC22D3, UBE2A, and/or ZBTB7B.
  • the marker gene is ATP5F1E, BUB3, CDK10, CENPB, COX7B, ELOB, FAM3A, IFIT1, JUND, NSMCE1, OSTF1, PDCD10, PRR13, QSOX1, RPL32, RPL35A, RPL36, RPLP1, RPN1, RPS15A, SERTAD3, SLC25A44, TSC22D3, UBE2A, ZBTB7B, EEF1A1, EIF4A3, HDAC6, HNRNPH2, IFFO1, INPP5D, POLR2F, and/or RPS19.
  • Table 1 Preferred marker genes, with database accession numbers and EMD values; EMD: earth movers distance (wasserstein distance), positive EMD values indicate larger UTR in ASD patients; SEQ ID NOs of exemplary 3'UTRs of the respective genes are indicated.
  • Gene NCBI Genbank Acc No EMD SEQ ID NO GeneID RPN1 6184 NM_002950.4 -0,3697 1 UBE2A 7319 NM_003336.4 -0,2578 2 MKRN2 23609 NM_014160.5 0,0134 3 UHMK1 63877 NM_175866.5 0,7158 4 IER3 8870 NM_003897.4 -0,1085 5 BUB3 7529 NM_004725.4 -0,5448 6 MLX 4691 NM_198204.2 -0,3364 7 CA2 760 NM_000067.3 -0,5689 8 ITM2A 9452 NM_004867.5 -0,4221 9 NT5
  • said term includes all sequences of a transcript, e.g. an mRNA, downstream of the open reading frame.
  • the 3'UTR preferably starts with the first nucleotide of the transcript 3' of the (last) stop codon of an open reading frame and ends with the last 3' nucleotide of the transcript. More preferably, the 3'UTR starts with the first nucleotide of the transcript 3' of the (last) stop codon of an open reading frame and ends with the last nucleotide 5' of the poly-A tail of the transcript.
  • Preferred 3'UTRs are those of SEQ ID NOs: 1 to 352, and, in a preferred embodiment, SEQ ID NOs: 2056 to 2063.
  • determining refers to semiquantitative or quantitative determination of an APA parameter referred to herein. Determining the value of an APA parameter may be carried out by any technique which allows for establishing a measure of quantity of an APA parameter in a semiquantitative or quantitative manner. Suitable techniques are in principle known in the art, e.g. from the references cited herein above and in the Examples, and are discussed elsewhere herein in more detail.
  • an APA parameter may be determined by any means deemed appropriate by the skilled person, e.g. for determining a length of a 3'UTR or a part thereof. Thus, 3'UTR or a part thereof may be sequenced, e.g.
  • a parameter of APA may also be determined by detecting the presence or absence of specific 3'UTR subsequences.
  • Preferred 3'UTR subsequences are disclosed herein as SEQ ID NOs:353 to 2048 and, in a preferred embodimennt, SEQ ID NOs:2064 to 2089.
  • the 3'UTR sequences shown in SEQ ID NOs:1 to 352 and 2056 to 2063 are exemplary sequences, since the corresponding gene products are subject to APA and, as a consequence, there is variation in 3'UTR sequences.
  • the subsequences of SEQ ID NOs:353 to 2048 and, optionally, 2064 to 2089 may or may not be comprised in the corresponding exemplary 3'UTR indicated, may be only comprised partially, may comprise additional sequences, and/or may comprise single nucleotide polymorphisms or other mutations.
  • determining the presence or an increased amount, compared to a healthy control, of 3'UTR subsequences designated by an indication including "ASD", in particular those of Table 12 and, in a preferred embodiment Table 15, is indicative of a subject suffering from ASD.
  • determining the presence or an increased amount, compared to a healthy control, of a nucleic acid sequence selected from the list consisting of SEQ ID NOs:353 to 1280 and/or, in a preferred embodiment SEQ ID NOs:2064 to 2074 is indicative of a subject suffering from ASD.
  • the subsequence determined preferably comprises at least 10, more preferably at least 12, even more preferably at least 14, contiguous nucleotides of any one of SEQ ID NO:1 to 2048 and/or optionally 2056 to 2089, in particular SEQ ID NOs:353 to 2048 and/or optionally 2064 to 2089.
  • an APA parameter may also be determined e.g. by classical hybridization methods, such as DNA/DNA blot or DNA/RNA blot methods, amplification of polynucleotides using subsequence-specific primers, and the like.
  • probe polynucleotides e.g.
  • a polynucleotide comprising a sequence of from 10, preferably from 12, more preferably from 14, still more preferably from 15, even more preferably from 16, to all contiguous nucleotides of any one of SEQ ID NOs:1 to 2048 and/or optionally from SEQ ID NOs: 2056 to 2089, or a reverse complement sequence thereof, is used as a probe polynucleotide and/or as a primer polynucleotide in said determination.
  • the term “reference”, as used herein, relates to a value, e.g. a value of an APA parameter, such as a 3'UTR length, or any value derived therefrom, e.g. a score, which can be correlated to the medical condition ASD and, preferably, which allows for the assessment of the invention to be made, more preferably enables allocation of a subject into either a group of subjects suffering from a disease or condition or being at risk for developing it, or a group of subjects which do not suffer from said disease or condition or which are not at risk for developing it.
  • Such a reference can be a threshold value, e.g. a threshold 3'UTR length, which separates these groups from each other.
  • a reference may in particular be derived from at least one reference group, the term "reference group" relating to a group of subjects with known status with regard to the assessment.
  • the reference group may e.g. be a group of subjects for which it is known whether they suffer from ASD.
  • the population of subjects in a reference group preferably comprises a plurality of subjects, e.g. at least 5, 10, 50, 100, 1,000, or 10,000 subjects.
  • the subject to be diagnosed and the subjects of the said reference group are of the same species.
  • the reference applicable for an individual subject may vary depending on various physiological parameters such as age, gender, or subpopulation.
  • a reference may be derived also from the average population. Assuming that contribution of actually afflicted subjects is low, such an average population reference group may be treated as a reference group known not to suffer from ASD; preferably, in such case, the size of the reference group is sufficiently large, e.g. at least 100, more preferably at least 1000, more preferably at least 10000 subjects.
  • a reference group may, in principle, also be a mixed population of subjects with regard to ASD, provided that the status of each member of said mixed population with regards to ASD is or becomes known before deriving a reference from such group.
  • comparing encompasses comparing the determined value of an APA parameter as referred to herein to a reference. It is to be understood that comparing as used herein refers to any kind of comparison made between the value determined with the reference. However, it is to be understood that, preferably, identical types of values are compared with each other, e.g., if an average length is determined, the reference shall also be an average length, if a relative length is determined, the reference shall also be a relative length, etc.
  • the term comparing also encompasses comparing a calculated score with a suitable reference score. The comparison may be carried out manually or computer assisted.
  • the value of the amount and the reference can be, e.g., compared to each other and the said comparison can be automatically carried out by a computer program executing an algorithm for the comparison.
  • the computer program carrying out the said evaluation will provide the desired assessment in a suitable output format.
  • the calculated score preferably combines information on the determinations in a multitude of marker genes.
  • the marker genes may be weighted in accordance with their contribution to the establishment of the differentiation, wherein the weighting factor of the individual marker genes may be different.
  • the score can be regarded as a classifier parameter for the assessing as set forth herein.
  • the method of assessing comprises step (a) determining a parameter of alternative polyadenylation (APA parameter) in said sample. Parameters of APA and methods for their determination have been described herein above. Preferably, the APA parameter is determined in a blood-derived sample as described herein above.
  • the parameter is determined as specified herein above; to the value obtained, standard mathematical and statistical operations may be applied, such as standardization, normalization, log- transformation, e.g. log10 transformation, and the like.
  • the method of assessing further comprises step (b) comparing the APA parameter determined in step (a) to a reference.
  • References have been described herein above. As the skilled person understands, in case determination is based on a multitude of marker genes, these are preferably compared to references and/or are included in calculating the score as well. As is also understood by the skilled person, values of parameters determined for marker genes are typically compared to corresponding references, as specified herein above.
  • an APA parameter determined for a marker gene X will be compared to a reference derived from measurements relating to said marker gene X.
  • a score is calculated from the amounts determined in step (a)
  • said score is compared to a reference score calculated from references based on the same marker gene(s) by the same mathematical operations.
  • the specific comparison made preferably depends on the specific reference(s) used.
  • the comparison may comprise determining whether the value of the sample in question is beyond said threshold; in case the reference is a reference range, the comparison may comprise establishing whether the value of the sample in question is within the reference range, or not.
  • the method of assessing further comprises step (c) based on the comparison in step (b), assessing said ASD.
  • the assessment in step (c) may in particular be based on the status of the reference group(s) and the reference derived therefrom.
  • a parameter of APA may be decreased or increased in an afflicted subject compared to a healthy reference.
  • a parameter value essentially identical to the reference preferably leads to an assessment of the subject under investigation being afflicted with the disease or condition, and a value different, in an embodiment significantly different, from said reference preferably leads to an assessment of the subject under investigation not being afflicted with the disease or condition.
  • a parameter value of a subject under investigation exceeds the aforesaid threshold reference toward the values of the reference group known to suffer from the disease or condition, it will be assumed that the subject suffers from the disease of condition; and in case a parameter value of a subject under investigation exceeds the aforesaid threshold toward the values of the reference group known to not suffer from the disease or condition, it will be assumed that the subject does not suffer from the disease or condition.
  • the present invention further relates to a detection polynucleotide comprising of from 10 to all contiguous nucleotides of a nucleic acid sequence present in the 3'UTR of a marker gene from a subject suffering from ASD but not in a subject not suffering from ASD; or present in the 3'UTR of a marker gene from a subject not suffering from ASD but not present in a subject suffering from ASD.
  • the detection polynucleotide is preferably derived from sequence differing between a subject suffering from ASD and a subject not suffering from ASD.
  • said detection polynucleotide comprises, preferably consists of, a nucleic acid sequence of any one of SEQ ID NOs:353 to 2048 and/or optionally 2064 to 2089, of a fragment comprising at least 10, more preferably at least 12, even more preferably at least 14, contiguous nucleotides thereof, as the only gene specific sequence of a marker gene specified in Table 1 or, in a preferred embodiment, Table 14.
  • the term polynucleotide has been specified herein above; in concurrence, the term “detection polynucleotide”, as used herein, refers to a linear or circular nucleic acid molecule having the specified properties.
  • the detection polynucleotides of the invention have the activity of specifically binding to a 3'UTR from a subject suffering from ASD, or specifically binding to a 3'UTR from a subject not suffering from ASD. Methods for testing whether a condidate detection polynucleotide has the aforesaid activity are known in the art. Unless specifically indicated otherwise, reference to specific detection polynucleotides herein preferably includes detection polynucleotide variants. A detection polynucleotide variant referred to herein has the activity as specified herein above.
  • the detection polynucleotide or variant thereof preferably comprises a sequence which is essentially the reverse complement of the sequence of a 3'UTR of a transcript of a marker gene referred to herein, preferable comprises a sequence which is the reverse complement of such transcript.
  • the present invention also relates to a means for determining an APA parameter for use in diagnosis; and to a use of means for determining an APA parameter for the manufacture of a diagnostic.
  • the present invention relates to a means for determining an APA parameter for use in diagnosing ASD; and to a use of a means for determining an APA parameter for the manufacture of a diagnostic for diagnosing ASD.
  • the term "means for determining an APA parameter" is understood by the skilled person in view of the description herein and in principle includes each and every means deemed suitable by the skilled person to determine an APA parameter as specified elsewhere herein.
  • said means is specific for determining an APA parameter.
  • the means is essential for determining an APA parameter, i.e. preferably is required to make a determination of an APA parameter possible.
  • the term preferably relates to means directly interacting with at least one 3'UTR of a marker gene as specified herein above.
  • the means for determining an APA parameter preferably is a polynucleotide comprising a nucleic acid sequence specifically hybridizing to a transcript of at least one of the marker genes specified in Table 1, more preferably specifically hybridizes to a 3'UTR of at least one of said genes.
  • the means for determining an APA parameter preferably is a probe polynucleotide or a primer polynucleotide, preferably comprising a sequence of at least 10 contiguous nucleotides of any one of SEQ ID NOs:1 to 2048, or, in a preferred embodiment, 2056 to 2089, or a reverse complement thereof, more preferably is a detection polynucleotide of the present invention.
  • the polynucleotide being a means for determining an APA parameter does not necessarily have to be specific for a sequence specific for a subject suffering from ASD or for a subject not suffering from ASD.
  • said means may be any means allowing such an APA parameter to be determined.
  • said means may e.g. be a primer polynucleotide enabling amplification and/or sequencing, and thereby length determination, of a 3'UTR of a marker gene referred to herein.
  • Said means may also be a probe polynucleotide, which may enable detection of a specific sequence, but may also enable length determination of a 3'UTR of a marker gene referred to herein, as is the case e.g. in traditional RNA/DNA blotting techniques ("Northern blotting").
  • diagnosis refers to assessing the probability according to which a subject is suffering from a disease or condition or is at risk of developing a disease or condition. Accordingly, the method may provide an aid for diagnosis since it might be necessary to further strengthen or confirm said diagnosis by, e.g., a medical practitioner.
  • such an assessment may not be correct for 100% of the subjects to be diagnosed.
  • the term preferably, requires that a statistically significant portion of subjects can be correctly identified as suffering from the disease and/or that a statistically significant portion of subjects can be correctly identified as not suffering from the disease, wherein correct identification preferably is verified based on a population of subjects with a clinical ASD diagnosis established according to applicable guidelines. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student ⁇ s t-test, Mann-Whitney test, Likelihood ratio test, etc.
  • Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
  • Preferred p-values are 0.1, 0.05, 0.01, 0.05, or 0.001. It will be understood, moreover, that the methods of the present invention essentially provide an aid for diagnosis and may be included into or supplemented by other diagnostic measures. Diagnosing according to the present invention includes confirmation and classification of the relevant disease or its symptoms. Confirmation relates to the strengthening or substantiating a diagnosis already established using other indicators or markers.
  • Classification relates to allocating the diagnosis according to the strength or kind of symptoms into different classes, such as those indicated herein above.
  • the present invention also relates to a database, preferably tangibly embedded on a data carrier, comprising an identifier of at least one of the marker genes specified in Table 1 and/or, in a preferred embodiment, Table 14, allocated to at least one APA parameter.
  • database refers to a collection of data which may be physically and/or logically grouped together. Accordingly, the database preferably comprises an allocation of at least one identifier of at least one of the marker genes specified in Table 1 to at least one APA parameter.
  • the APA parameter preferably is a reference as specified herein above.
  • the APA parameter may, however, also be an APA parameter value, e.g. of a subject of known ASD status; in accordance, in particular in case the database comprises APA parameter values from a multitude of non-identical subjects with known ASD status, a reference may be calculated from such APA parameter values. Accordingly, the database preferably further comprises, allocated to the APA parameter, an identifier of a diagnosis allocated to said APA parameter value or reference. Thus, the database enables allocating a marker gene to an APA parameter value, and, preferably, allocating an APA parameter value determined in a sample to a (tentative) diagnosis. As the skilled person understands from the description herein above, the reference may be a score, so the APA parameter comprised in the database may also be said score.
  • the database preferably comprises further data, such as upper and/or lower detection limits, references for further marker genes, in particular those described herein above, data relevant for plausibility checks, and the like.
  • the database comprises data on one or more determining methods to use, lot-specific data, e.g. for means for determination of an APA parameter, and the like.
  • the database may be implemented in a single data storage medium or in physically separated data storage media being operatively linked to each other.
  • the database comprises a data collection on a suitable storage medium, preferably tangibly embedded thereon.
  • the database preferably further comprises a database management system.
  • the database management system preferably is a network-based, hierarchical or object-oriented database management system.
  • the database may be a federal or integrated database.
  • the database will be implemented as a distributed (federal) system, e.g. as a Client-Server- System.
  • the database is structured as to allow a search algorithm to compare a test data set with the data sets, in particular references, comprised by the data collection. Specifically, by using such an algorithm, the database can be searched for similar or identical data sets being indicative for a subpopulation or effect as set forth above (e.g. a query search).
  • a data set fulfilling the comparison criteria as detailed elsewhere herein can be identified in the database, the test data set will be associated with the diagnosis.
  • the present invention further relates to a data carrier comprising the database a referred to herein above.
  • a data carrier comprising the database a referred to herein above.
  • the term "data carrier” is used in a wide sense and includes any device adapted for carrying data.
  • the data carrier may be human readable and/or machine readable.
  • the data carrier may be a printed information comprising the database.
  • the data carrier is a device carrying data in a machine-readable form e.g. as a CD, DVD, memory device or memory unit of a device specified herein, mass storage unit, and the like.
  • the present invention also relates to a device comprising a means for determining an APA parameter according to the present invention, a database according to the present invention and/or a data carrier according to the present invention.
  • the term "device” includes any and all apparatuses comprising the components specified.
  • the device is adapted to perform a method as specified herein, in particular an assessment method, so the device may e.g. be a diagnostic device.
  • the device preferably comprises (i) an analysis unit comprising a means for determining an APA parameter in a sample, and, operatively connected thereto (ii) an evaluation unit comprising tangibly embedded executable instructions for performing a method as specified herein.
  • the present invention relates to a use of a polynucleotide according to the present invention an oligonucleotide according to the present invention, a database according to the present invention, a data carrier according to the present invention, a device according to the present invention, and/or a kit according to the present invention, for diagnosing, treating, and/or preventing an ASD.
  • the present invention relates to a method of providing at least one marker gene for use in the method of assessing an ASD of the present invention, the method comprising (I) providing 3'UTR sequencing read starting sites of a multitude of gene transcripts in samples of a multitude subjects from a reference group, wherein a fraction of said multitude of subjects is suffering from ASD; (II) allocating the 3'UTR sequencing read starting sites of step (I) to reference 3'UTR coordinates and determining 3'UTR lengths (transcript 3'UTR lengths); (III) modeling the distribution of transcript 3'UTR lengths in a multivariate probability distribution, preferably a Dirichlet-Multinomial distribution; and (IV) identifying genes with 3'UTR length differences between ASD and non-ASD samples via a Likelihood Ratio test, thereby providing at least one marker gene for use in the method of assessing an ASD.
  • the method of providing is an in vitro method and may comprise steps in addition to those specifically referred to.
  • step (III) may be followed by step (V) ranking the genes identified in step (IV) via Jensen-Shannon distance, or Wasserstein distance between ASD and non- ASD samples; also, the method may further comprise step (VI) selecting at least one gene with a permutation-corrected p-value from step (IV) below 0.01 and preferably, an absolute distance from step (V) above the 50% percentile, preferably the 75% percentile, more preferably the 85% percentile.
  • the method comprises at least one further feature as specified herein in the Examples.
  • the method of providing may also be used to provide one or more reference(s) for an APA parameter.
  • the method may also be a method for providing at least one APA parameter for use in the method of assessing an ASD of the present invention, or a method for providing at least one marker gene and an allocated APA parameter for use in the method of assessing an ASD of the present invention.
  • the term "providing” is used herein in a broad sense including any and all means and methods of making the indicated information or item available.
  • a 3'UTR sequencing read starting site may be provided as an information, preferably tangibly embedded on a data carrier.
  • the 3'UTR sequencing read starting site may be known as such to the skilled person, or may be newly identified, preferably as specified herein in the Examples.
  • providing a 3'UTR sequencing read starting site may be identifying, preferably experimentally identifying, the 3'UTR sequencing read starting site.
  • a multitude of 3'UTR sequencing read starting sites is provided.
  • at least one 3'UTR sequencing read starting site is provided per marker gene.
  • the statistical methods referred to herein, in particular including Dirichlet-Multinomial distribution, Likelihood Ratio test, Jensen-Shannon distance, and Wasserstein distance calculations are textbook knowledge available to the skilled person.
  • Embodiment 1 A method for assessing an autism spectrum disorder (ASD) in a sample from a subject, the method comprising (a) determining a parameter of alternative polyadenylation (APA parameter) in said sample; (b) comparing the APA parameter determined in step (a) to a reference, and (c) based on the comparison in step (b), assessing said ASD.
  • Embodiment 2 The method of embodiment 1, wherein said sample is a non-brain sample.
  • Embodiment 3 The method of embodiment 1 or 2, wherein said sample is a sample of a bodily fluid.
  • Embodiment 4 The method of any one of embodiments 1 to 3, wherein said sample is a sample comprising cells.
  • Embodiment 5 The method of any one of embodiments 1 to 4, wherein said sample is a blood sample or a blood-derived sample.
  • Embodiment 6 The method of any one of embodiments 1 to 6, wherein said APA parameter comprises an mRNA 3' untranslated region (3'UTR) length of at least one marker gene selected from the list consisting of RPN1, UBE2A, MKRN2, UHMK1, IER3, BUB3, MLX, CA2, ITM2A, NT5C3A, FGR, M6PR, AK2, IFRD1, RHBDF1, MMP25, CALCOCO1, NUB1, IFNGR1, TRIT1, OFD1, VAMP3, GDI2, ATP11B, MRPS35, CASP8, ZBTB11, ISOC1, SMG6, FGF22, DGCR2, CAMK2A, RPL31, TRIB2, FAM3A, RAB7A, LXN, KEAP1, PCNP, EPB41L3, ATRX, SLC25A
  • Embodiment 7 The method of embodiment 7, wherein said APA parameter comprises mRNA 3'UTR lengths of at least two, preferably at least three, more preferably at least four, even more preferably at least five, marker genes selected from said list.
  • Embodiment 8 The method of any one of embodiments 1 to 7, wherein said APA parameter comprises mRNA 3'UTR lengths of at least ten preferably at least 25, marker genes selected from said list.
  • Embodiment 9 The method of any one of embodiments 1 to 8, wherein said APA parameter comprises at least one mRNA 3'UTR length of RPN1, UBE2A and/or MKRN2.
  • Embodiment 10 The method of any one of embodiments 1 to 9, wherein said APA parameter comprises mRNA 3'UTR lengths of RPN1, UBE2A, and MKRN2.
  • Embodiment 11 The method of any one of embodiments 1 to 10, wherein said APA parameter comprises mRNA 3'UTR lengths of RPN1, UBE2A, MKRN2, UHMK1, IER3, BUB3, MLX, CA2, ITM2A, and/or NT5C3A.
  • Embodiment 12 The method of any one of embodiments 1 to 11, wherein said APA parameter comprises mRNA 3'UTR lengths of at least two, preferably at least three, more preferably at least four, even more preferably at least five, still more preferably at least six, still more preferably at least seven, still more preferably at least eight, still more preferably at least nine, most preferably all ten, marker genes selected from the list consisting of RPN1, UBE2A, MKRN2, UHMK1, IER3, BUB3, MLX, CA2, ITM2A, and NT5C3A.
  • said APA parameter comprises mRNA 3'UTR lengths of at least two, preferably at least three, more preferably at least four, even more preferably at least five, still more preferably at least six, still more preferably at least seven, still more preferably at least eight, still more preferably at least nine, most preferably all ten, marker genes selected from the list consisting of RPN1, UBE2A, MKRN2, UHMK1,
  • Embodiment 13 The method of any one of embodiments 1 to 12, wherein said APA parameter comprises mRNA 3'UTR lengths of the marker genes RPN1, UBE2A, MKRN2, UHMK1, IER3, BUB3, MLX, CA2, ITM2A, and NT5C3A.
  • Embodiment 14 The method of any one of embodiments 1 to 7, wherein said APA parameter comprises mRNA 3'UTR lengths of all of said marker genes.
  • Embodiment 15 The method of any one of embodiments 1 to 14, wherein said APA parameter is determined from 3' mRNA reads.
  • Embodiment 16 The method of any one of embodiments 1 to 15, wherein a polynucleotide comprising the reverse complement sequence of from 10 to all contiguous nucleotides of any one of SEQ ID NOs:1 to 352 and, optionally 2056 to 2063, is used as a probe polynucleotide and/or as a primer polynucleotide.
  • Embodiment 17 A detection polynucleotide comprising a sequence of from 10 to all contiguous nucleotides of any one of SEQ ID NOs:1 to 2048 and, optionally 2056 to 2089, or a reverse complement thereof, as the only gene specific sequence of the genes specified in any one of embodiments 7 to 13.
  • Embodiment 18 A means for determining an APA parameter for use in diagnosis.
  • Embodiment 19 Use of means for determining an APA parameter for the manufacture of a diagnostic.
  • Embodiment 20 A means for determining an APA parameter for use in diagnosing ASD.
  • Embodiment 21 Use of a means for determining an APA parameter for the manufacture of a diagnostic for diagnosing ASD.
  • Embodiment 22 The subject matter of any one of embodiments 18 to 21, wherein said means for determining an APA parameter is a polynucleotide comprising a nucleic acid sequence specifically hybridizing to a marker gene specified in Table 1.
  • Embodiment 23 The subject matter of any one of embodiments 18 to 22, wherein said polynucleotide specifically hybridizes to a 3'UTR of at least one marker gene specified in any one of embodiments 6 to 13.
  • Embodiment 24 The subject matter of any one of embodiments 18 to 23, wherein said polynucleotide is a probe polynucleotide.
  • Embodiment 25 The subject matter of any one of embodiments 18 to 24, wherein said polynucleotide is primer polynucleotide.
  • Embodiment 26 A database, preferably tangibly embedded on a data carrier, comprising an identifier of at least one of the marker genes specified in embodiment any one of embodiments 6 to 13 allocated to at least one APA parameter.
  • Embodiment 27 The database of embodiment 26, wherein said identifier is allocated to at least one APA parameter value derived from an apparently healthy subject and/or to at least one APA parameter value derived from at least one subject known to suffer from ASD.
  • Embodiment 28 The database of embodiment 26 or 27, wherein said APA parameter is a reference.
  • Embodiment 29 A data carrier comprising the database of any one of embodiments 24 to 27.
  • Embodiment 30 A device comprising a means for determining an APA parameter, a database according to any one of embodiments 25 to 28 and/or a data carrier according to embodiment 29.
  • Embodiment 31 A kit comprising at least one detection polynucleotide according to embodiment 17, preferably comprised in a housing.
  • Embodiment 32 The kit of embodiment 31, further comprising a data carrier according to embodiment 28.
  • Embodiment 33 A method for treating and/or preventing an ASD in a subject, said method comprising (A) diagnosing or having diagnosed said ASD in said subject, and (B) treating and/or preventing said ASD in said subject.
  • Embodiment 34 A method for identifying a subject susceptible for treating and/or preventing an ASD, said method comprising diagnosing said ASD in said subject, and, thereby, identifying a subject susceptible for treating and/or preventing said ASD.
  • Embodiment 35 The method of embodiment 33 or 34, wherein said diagnosing comprises the steps of the method of any one of embodiments 1 to 16.
  • Embodiment 36 Use of a detection polynucleotide according to embodiment 17, a database according to any one of embodiments 26 to 28, a data carrier according to embodiment 29, a device according to embodiment 30, and/or a kit according to embodiment 31 or 32, for diagnosing, treating, and/or preventing an ASD.
  • Embodiment 37 The use of embodiment 36, wherein said use is for diagnosing and wherein said diagnosing is in vitro diagnosing.
  • Embodiment 38 A method of providing at least one marker gene for use in the method of assessing an ASD of the present invention, the method comprising (I) providing 3'UTR sequencing read starting sites of a multitude of gene transcripts in samples of a multitude subjects from a reference group, wherein a fraction of said multitude of subjects is suffering from ASD; (II) allocating the 3'UTR sequencing read starting sites of step (I) to reference 3'UTR coordinates and determining 3'UTR lengths (transcript 3'UTR lengths); (III) modeling the distribution of transcript 3'UTR lengths in a multivariate probability distribution, preferably a Dirichlet-Multinomial distribution; and (IV) identifying genes with 3'UTR length differences between ASD and non-ASD samples via a Likelihood Ratio test, thereby providing at least one marker gene for use in the method of assessing an ASD.
  • Embodiment 39 The method of embodiment 38 further having a feature of one of embodiments 1 to 37.
  • Embodiment 40 The subject matter of any one of embodiments 1 to 39, wherein said APA parameter comprises an mRNA 3' untranslated region (3'UTR) length of at least one marker gene selected from the list consisting of ATP5F1E, BUB3, CDK10, CENPB, COX7B, ELOB, FAM3A, IFIT1, JUND, NSMCE1, OSTF1, PDCD10, PRR13, QSOX1, RPL32, RPL35A, RPL36, RPLP1, RPN1, RPS15A, SERTAD3, SLC25A44, TSC22D3, UBE2A, and/or ZBTB7B, preferably the list consisting of ATP5F1E, BUB3, CDK10, CENPB, COX7B, ELOB, FAM3A, IFIT1, JUND, NSMCE1, OSTF1, PDCD10, PRR13
  • Embodiment 41 The subject mater of any one of embodiments 1 to 40, wherein said APA parameter comprises an mRNA 3' untranslated region (3'UTR) length of at least five, preferably at least ten, more preferably at least 15, still more preferably at least 20, even more preferably at least 25, most preferably all, marker gene selected from the list consisting of ATP5F1E, BUB3, CDK10, CENPB, COX7B, ELOB, FAM3A, IFIT1, JUND, NSMCE1, OSTF1, PDCD10, PRR13, QSOX1, RPL32, RPL35A, RPL36, RPLP1, RPN1, RPS15A, SERTAD3, SLC25A44, TSC22D3, UBE2A, and/or ZBTB7B, preferably the list consisting of ATP5F1E, BUB3, CDK10, CENPB, COX7B, ELOB, FAM3A, IFIT1, JUND, NSMCE1, OSTF1,
  • Embodiment 42 The subject matter of any one of embodiments 1 to 41, wherein assessing ASD is assessing ASD level 1. All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.
  • Figure Legends Fig.1 Experimental setup to study APA from blood of ASD patients and healthy controls.
  • A Overview of samples used for analysis of APA differences in blood as a diagnostic tool for ASD.
  • B Schematic representation of SPRI bead- based size selection before and after cDNA amplification to remove contaminating RBC transcripts (HBB & HBA1/2) that are smaller in fragment size than most of the PBMC cDNA.
  • the RT-mix was as follows: Table 3: RT mix for cDNA preparation of all samples Once the 10 ⁇ l RT reaction is set up the following program was run on the thermocycler: Table 4: Thermocycler cycling conditions for reverse transcription After the RT reaction the cDNA was amplified to have enough material for the tagmentation protocol. The following reaction composition was used: Table 5: cDNA amplification PCR reaction mix After setting the PCR reaction the following PCR program was set up. Table 6: cDNA Amplification PCR program Post PCR amplification, from fresh whole blood RNA samples, two rounds of a 0.8X SPRI bead purification was performed after the RT reaction to avoid primer carry over into the cDNA.
  • Table 7 Tagmentation reactionmix for 3‘UTR library prep Immediately after the tagmentation, 1.25 ⁇ l of 0.2 % SDS was added to the reaction and incubated at RT for 5 mins to strip off the transposase. The normal gap filling step after tagmentation was avoided in order to enrich specifically for the 3’ ends of the mRNA transcripts (Fig.2). In order to amplify the tagmented product the following reaction was set up (Table 8) with using the PCR program described in Table 9.
  • Table 8 Library PCR reaction mix post tagmentation of PBMC cDNA
  • Table 9 Library PCR program to amplify final 3’UTR libraries Finally, two rounds of 0.7X bead purification was performed and the final 3’UTR libraries were eluted into 11 ⁇ l of nuclease-free water. The library concentrations were determined using qubit DNA high sensitivity kit (1 ⁇ l) and then the average library peak size was determined by running 1 ⁇ l of library on the bioanalyzer. The equimolar volumes of samples were pooled for sequencing using a combination of unique dual indexed libraries (i7 + sample barcode).
  • the final libraries were run on the NextSeq2000 sequencing platform using a P3200 cycles kit with paired end sequencing (130+8+0+100; R1+i7+i5+R2).
  • the amount of the library loaded on to the sequencer was 1100 pM (with 14% PhiX spike-in) and using a custom polyA-seq primer (Hennig et al., (2016) G3 Genes
  • the read2 was long enough (100bp) to go through the poly(A) tail into the ends of the 3’UTR thus allowing to identify true 3’UTR ends.
  • Example 2 Method usage for Diagnosis To show an application of the method described herein, we analyzed a blood sample from a male child clinically diagnosed with ASD. The child was diagnosed with the following co- morbidities: dysmorphic traits, language disabilities, learning disabilities, macrocephaly, and mobility issues.
  • Example 2 Sample preparation To derive an ASD diagnosis for an individual via RNA-sequencing, the blood was collected as described in Example 1, section 1.1., and cDNA preparation and RBC transcript depletion was performed as described in Example 1, sections 1.2 and 1.3, respectively, Then, the sample was tagmented and sequenced as described in Example 1, section 1.4. 2.23’UTR size determination The sequenced sample’s information, in the form of a fastq file containing both reads’ nucleotide sequences, was aligned to the Human genome (GRCh38 assembly) using STAR. In total, 4.226.937 reads were sequenced, with 68% of those uniquely mapped to the genome.
  • the read 1 was used to map the 3’ sequenced reads onto genomic coordinates and the 3’UTR regions were identified using Ensembl annotations. From these coordinates, each transcript’s UTR size was calculated. Next, these raw transcript UTR sizes were assigned to the nearest reference UTR polyadenylation peak, obtained in Example 1, generating a matrix with transcript counts per peak for the analyzed sample. 2.3 Diagnosis prediction Finally, the relative counts of each peak were calculated in relation to the total counts per UTR (Table 11) and this matrix was used as input for the ASD classification model obtained and described in Example 1. The sample was predicted by the logistic regression model to be from an individual with ASD, with a probability of 89.7%, confirming the previous behavioral diagnosis.
  • Table 11 Proportion of peak counts per UTR for a selected group of 3 UTRs.
  • Example 3 RNA-sequencing-free methods for diagnosis To enable methods of diagnosis that are not reliant on RNA-sequencing technology, and are thus more applicable to procedures already present in most clinics that work hand-in-hand with medical doctors, the sequences described in SEQ ID NOs:1 to 2048, in particular SEQ ID NOs:353 to 2048, are used to produce oligonucleotide probes, e.g. DNA oligonucleotide probes, specifically hybridizing to the aforesaid sequences.
  • oligonucleotide probes e.g. DNA oligonucleotide probes
  • the probes derived from the sequences designated by an indication including "ASD” are more prevalent in samples from individuals with ASD, and those derived from the sequences designated by an indication including "CTRL” are more frequent in healthy individuals.
  • This can be used to assess an ASD spectrum disorder by any hybridization method deemed appropriate by the skilled person by methods in principle known to the skilled person.
  • multiple probes are immobilized on a chip for RNA analysis such as microarrays, e.g. as proposed by Schena M, et al. (1995) Science 270(5235):467.
  • an RNA extract from the patient is fluorescently labeled and loaded to the microarray.
  • the immobilized probes hybridize with the sample RNA molecules, thus resulting in an increase of fluorescence intensity over a background level, which can be measured using a fluorescent scanner.
  • the relative flourescence of each probe can be obtained by dividing the probe’s fluorescence intensity by the sum of intensity of all probes for each UTR. This procedure generates a matrix comparable to the one described in Example 2, section 2.3. which can then be used as input for the ASD classification model obtained and described in Example 1, generating a binary and/or probability-based ASD diagnosis prediction.
  • oligonucleotide probes hybridizing specifically to at least one sequence from Table 12 or Table 13, and/or optionally Table 15 and/or Table 16 are labeled in a detectable manner.
  • Specific binding of a probe hybridizing to a sequence of Table 12, or optionally Table 15, is indicative of ASD, while specific binding of a probe of Table 13, or optionally Table 16, is indicative of non-ASD.
  • a multitude of oligonucleotide probes specifically hybridizing to a multitude of nucleic acid sequences of Table 12, and optionally Table 15, can be labeled with a first color, e.g. green, and/or a multitude of oligonucleotide probes specifically hybridizing to a multitude of nucleic acid sequences of Table 13, and optionally Table 16, can be labeled with a second color, e.g. red.
  • oligonucleotide probes are hybridized to RNA or cDNA prepared from a sample of a subject, and the resulting color intensity caused by bound oligonucleotide probes or their ratio is used as a parameter to asses ASD.
  • length of UTRs and/or presence of sequences from Table 12 and/or Table 13, and/or optionally Table 15 and/or Table 16 is determined by qRT-PCR by methods known to the skilled person.
  • Table 12 ASD subsequences of marker FAM3A 451 - 452 genes RAB7A_UTR5 453 - 458 marker gene ASD LXN_UTR2 459 subsequence(s), KEAP1 460 SEQ ID NOs PCNP_UTR4 461 - 469 RPN1 353 - 355 EPB41L3 470 - 473 UBE2A_UTR4 356 ATRX_UTR24 474 MKRN2_UTR6 357 - 359 SLC25A24 475 - 476 UHMK1 360 - 365 RBM22 477 - 479 IER3 366 FKBP1A 480 - 482 BUB3 367 - 371 SNX5_UTR3 483 MLX 374 PABPC4 484 CA2_UTR6 375 - 380 TINF2 485 ITM2A 381 COMT_UTR3 486 - 488 NT5C3A 382

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Abstract

La présente invention concerne une méthode d'évaluation d'un trouble du spectre autistique (TSA) dans un échantillon provenant d'un sujet, la méthode consistant (a) à déterminer un paramètre de polyadénylation alternative (paramètre PAA) dans ledit échantillon, (b) à comparer le paramètre PAA déterminé à l'étape (a) avec une référence, et (c) à évaluer ledit TSA sur la base de la comparaison effectuée dans l'étape (b). La présente invention concerne également des polynucléotides, des moyens pour déterminer un paramètre PAA destiné à être utilisé à des fins diagnostiques, des bases de données, des dispositifs et d'autres méthodes associées.
PCT/EP2024/078720 2023-10-13 2024-10-11 Évaluation d'un trouble du spectre autistique par analyse de polyadénylation alternative Pending WO2025078620A1 (fr)

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CN120330324A (zh) * 2025-06-20 2025-07-18 广西医科大学 一种基于泛凋亡相关基因的孤独症谱系障碍诊断试剂盒

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120330324A (zh) * 2025-06-20 2025-07-18 广西医科大学 一种基于泛凋亡相关基因的孤独症谱系障碍诊断试剂盒

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