WO2020169635A1 - Procédé pour déterminer l'incertitude du niveau de mosaïcisme placentaire d'un échantillon dans un dépistage prénatal non invasif - Google Patents

Procédé pour déterminer l'incertitude du niveau de mosaïcisme placentaire d'un échantillon dans un dépistage prénatal non invasif Download PDF

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WO2020169635A1
WO2020169635A1 PCT/EP2020/054287 EP2020054287W WO2020169635A1 WO 2020169635 A1 WO2020169635 A1 WO 2020169635A1 EP 2020054287 W EP2020054287 W EP 2020054287W WO 2020169635 A1 WO2020169635 A1 WO 2020169635A1
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mosaicism
chromosome
sample
degree
trisomic
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PCT/EP2020/054287
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English (en)
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Marian Grendár
Dušan Loderer
Zora Lasabová
Ján Danko
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Grendar Marian
Dušan Loderer
Lasabova Zora
Danko Jan
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Publication of WO2020169635A1 publication Critical patent/WO2020169635A1/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
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/10—Ploidy or copy number detection
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/20—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems

Definitions

  • the present invention relates to Non-lnvasive Prenatal Screening (NIPS). Particularly, the present invention relates to methods for determining the uncertainty of the degree of placental mosaicisms in a sample obtained from a pregnant subject. The present invention further relates to methods for determining whether or not a sample qualifies for determination of the degree of placental mosaicism. In accordance with the present invention, the related width of the monosomic, trisomic, and tetrasomic mosaicism intervals are used for determining the degree of monosomic, trisomic, and tetrasomic (placental) mosaicism within the read -counting approach to NIPS.
  • NIPS Non-lnvasive Prenatal Screening
  • NIPT Noninvasive Prenatal Testing
  • WGS Whole Genome Sequencing
  • CNV Copy Number Variation
  • Non-lnvasive Prenatal Screening is a liquid biopsy of the placenta (Harasim and Wagner, Noninvasive Prenatal Testing (NIPT), 67-82, (2016)).
  • the basic objective of NIPS is the determination of the aneuploid status of a placenta (Poot, Molecular Syndromology 6(4), 153- 155 (2015)); i.e., deciding, whether it is euploid or aneuploid in a chromosome.
  • NIPS places a sample into one of these categories: monosomic, disomic, trisomic, or tetrasomic.
  • the conventional NIPS places it into one of the categories (monosomy, disomy, trisomy, tetrasomy) and hence, the placenta is misdiagnosed.
  • the misdiagnosis may be harmful. It is thus of interest to develop a method for measuring the degree of mosaicism, e.g., placental mosaicism.
  • the present invention it was an objective to measure and quantify the degree of mosaicism of a given sample (e.g., pregnant/maternal; placental; fetal subject) in a suitable screening method, e.g., NIPS.
  • a suitable screening method e.g., NIPS.
  • objectives comprise provision of a quality control (QC) criterion for determination of the degree of (preferably fetal) mosaicism, e.g., placental mosaicism, in a suitable screening method such as, e.g., NIPS; determination of the suitability of a given sample (e.g., from pregnant/maternal subject; placental; fetal subject) for determining the degree of (preferably fetal) mosaicism, e.g., placental mosaicism, in a suitable screening method such as, e.g., NIPS; or assessment of the suitability of an implementation of suitable screening methods such as, e.g., NIPS for determining the degree of (preferably fetal) mosaicism (e.g., placental mosaicism).
  • the NIPS may be calibrated as also described and provided herein in context with the present invention.
  • placental mosaicism preferably placental mosaicism is meant or determined in accordance with the present invention.
  • the present invention relates to methods for determining the degree of (preferably fetal) monosomic, trisomic, and tetrasomic mosaicism, e.g., placental mosaicism, based on the respective intervals of mosaicism.
  • the related width of the monosomic, trisomic, and tetrasomic mosaicism intervals are used for determining the degree of monosomic, trisomic, and tetrasomic (e.g., placental) mosaicism within the read-counting approach to NIPS.
  • fetal (e.g., placental) mosaicism can be determined as described and provided herein.
  • the maternal trisomic and/or monosomic mosaicism is of known degree m o
  • the maternal trisomic and/or monosomic mosaicism degree can be 0 (i.e. the mother can be euploid).
  • a sample for use in the methods of the present invention e.g. from a mother or pregnant female subject is assumed to be to be euploid.
  • the methods of the present invention enable to determine whether a sample, e.g. from a mother or pregnant female subject may be a mosaicism, e.g. trisomic or tetrasomic.
  • the present invention relates to a method for determining the uncertainty of the degree of trisomic (preferably placental) mosaicism in a sample with the maternal trisomic mosaicism of the degree by the trisomic placental mosaicism interval
  • chromosome or chromosome representation
  • the maternal chromosome representation may be composed of two components: (1- ) is the euploid contribution, and 1.5 is the trisomic one.
  • the placental chromosome representation may comprise the euploid component (1- ) and the trisomic one: 1.5 . Assuming fetal fraction is , the chromosome representation of a sample would be
  • the band may be defined as the set of all ( , ) such that the following two conditions are satisfied: £ (1 + 0.5 ) + 0.5 ( - )
  • the degree g of the placental trisomic mosaicism of a sample with the chromosome representation and fetal fraction may lay in the interval [ min’ max ], where
  • the present invention relates to a method for determining whether or not a sample characterized by its fetal fraction , the read representation of the chromosome , maternal trisomic mosaicism of the degree and the width of trisomic mosaicism interval as defined herein above qualifies for determination of the degree of trisomic (preferably placental) mosaicism with the uncertainty at most ', comprising determining whether ⁇ ',
  • the present invention relates to a method as defined herein above, wherein in a sample with the maternal trisomic mosaicism the trisomic (preferably placental) mosaicism concerns any autosome or sex chromosome (preferably X).
  • the lower and upper bounds of the interval can be more conveniently expressed in terms of the z-score instead of .
  • the present invention relates to a method for determining whether or not a sample from a mother euploid at a chromosome qualifies for determination of the degree of trisomic (preferably placental) mosaicism at the chromosome with the uncertainty at most ' is equivalent to determining whether
  • ⁇ is the read representation of the chromosome of the sample
  • ⁇ is fetal fraction of the sample
  • ⁇ is the minimal value of the read representation of the chromosome in a set of euploid samples
  • ⁇ is the maximal value of the read representation of the chromosome in a set of euploid samples
  • ⁇ is the read representation of the chromosome of the sample
  • ⁇ is fetal fraction of the sample
  • ⁇ is the minimal value of the read representation of the chromosome in a set of euploid samples
  • ⁇ is the maximal value of the read representation of the chromosome in a set of euploid samples
  • the maternal chromosome representation may be composed of two components: ( 1 - ) is the euploid contribution, and 1.5 - is the trisomic one.
  • the placental chromosome representation may comprise the euploid component
  • the chromosome representation of a sample may be
  • the band may be defined as the set of all ( , ) such that the following two conditions are satisfied: £ ( 1 + 0.5 ⁇ ) - 0.5 ( + ) and
  • the degree of the placental monosomic mosaicism of a sample with the chromosome representation and fetal fraction may lay in the interval [ min’ max ] , where
  • the present invention relates to a method for determining whether or not a sample characterized by its fetal fraction , the read representation of the chromosome , the maternal trisomic mosaicism of the degree and the width of monosomic (preferably placental) mosaicism interval as defined herein above qualifies for determination of the degree of monosomic mosaicism with the uncertainty at most comprising determining whether ⁇ wherein if 3 ' said sample is not qualified for determining the degree of monosomic mosaicism with uncertainty at most or
  • the present invention relates to a method as defined herein above, wherein the monosomic (preferably placental) mosaicism concerns any autosome or sex chromosome (preferably X).
  • the lower and upper bounds of the interval can be more conveniently expressed in terms of the z-score instead of .
  • the present invention relates to a method for determining whether or not a sample qualifies for determination of the degree of monosomic mosaicism with the uncertainty at most ' is equivalent to determining whether
  • ⁇ is the read representation of the chromosome of the sample
  • ⁇ is fetal fraction of the sample
  • ⁇ is the minimal value of the read representation of the chromosome in a set of euploid samples
  • ⁇ is the maximal value of the read representation of the chromosome in a set of euploid samples
  • the present invention further relates to a method for determining the uncertainty of the degree of trisomic (preferably placental) mosaicism in a sample with the maternal monosomic mosaicism of the degree ' by the trisomic placental mosaicism interval [ ' min’ 'max] with the width max min where (0.5 ⁇ ( - 1 ) + 1 )
  • the maternal chromosome representation may be composed of two components: ( 1 - ') is the euploid contribution, and 0.5 - is the monosomic one.
  • the placental chromosome representation may comprise the euploid component
  • the chromosome representation of a sample may be
  • the band may be defined as the set of all ( , ) such that the following two conditions are satisfied:
  • the degree ' of the placental trisomic mosaicism of a sample with the chromosome representation and fetal fraction lays in the interval [ ' , ], where (0.5 ⁇ ( - 1)+ 1)
  • the present invention relates to a method for determining whether or not a sample characterized by its fetal fraction , the read representation of the chromosome , maternal monosomic mosaicism of the degree ' and the width of trisomic mosaicism interval as defined herein above qualifies for determination of the degree of trisomic (preferably placental)
  • the present invention relates to a method as defined herein above, wherein the monosomic (preferably placental) mosaicism concerns any autosome or sex chromosome (preferably X).
  • the lower and upper bounds of the interval can be more conveniently expressed in terms of the z-score instead of . Accordingly, in one embodiment of the present invention, the read representation of a chromosome is replaced by the z-score
  • ⁇ is the read representation of the chromosome of the sample
  • ⁇ is fetal fraction of the sample
  • ⁇ is the minimal value of the read representation of the chromosome in a set of euploid samples
  • ⁇ is the maximal value of the read representation of the chromosome in a set of euploid samples
  • the maternal chromosome representation may be composed of two components: ( 1 - ') is the euploid contribution, and 0.5 ' is the monosomic one.
  • the placental chromosome representation comprises the euploid component
  • the band may be defined as the set of all ( , ) such that the following two conditions are satisfied: £ (1- 0.5 ') + 0.5 ( ') and
  • the degree of the placental monosomic mosaicism of a sample with the chromosome representation and fetal fraction may lay in the interval
  • the present invention relates to a method for determining whether or not a sample characterized by its fetal fraction , the read representation of the chromosome , the maternal monosomic mosaicism of the degree ' and the width of monosomic (preferably placental) mosaicism interval as defined herein above qualifies for determination of the degree of monosomic mosaicism with the uncertainty at most comprising determining whether
  • the present invention relates to a method as defined herein above, wherein the monosomic (preferably placental) mosaicism concerns any autosome or sex chromosome (preferably X).
  • the lower and upper bounds of the interval can be more conveniently expressed in terms of the z-score instead of . Accordingly, in one embodiment of the present invention, the read representation of a chromosome is replaced by the z-score
  • the (maternal) sample may be any sample of a pregnant subject (e.g., mammal, preferably human) comprising nucleic acid molecules (e.g., DNA or RNA, preferably DNA) or fragments or segments thereof, e.g., of placental or fetal origin (e.g., circulating fetal DNA or cfDNA).
  • a pregnant subject e.g., mammal, preferably human
  • nucleic acid molecules e.g., DNA or RNA, preferably DNA
  • fragments or segments thereof e.g., of placental or fetal origin (e.g., circulating fetal DNA or cfDNA).
  • placental or fetal origin e.g., circulating fetal DNA or cfDNA
  • Examples may comprise inter alia blood, particularly plasma or serum (preferably plasma), preferably derived from the placenta.
  • the preparation of serum or plasma from the maternal blood sample may be carried out by standard techniques known in the art.
  • Serum and plasma nucleic acid extraction methods allowing the purification of DNA or RNA from larger volumes of maternal sample increase the amount of fetal nucleic acid material for analysis and thus improve the accuracy.
  • a sequence-based enrichment method could also be used on the maternal serum or plasma to specifically enrich for fetal nucleic acid sequences.
  • the pregnant subject is mammal, preferably a female human subject.
  • cfDNA is circulating in maternal blood and can be measured by several sequencing methods, particularly MPS (massive parallel sequencing) methods as known in the art and also described herein.
  • the cfDNA also comprises nucleic acid (particularly DNA) molecules from the fetus of a pregnant subject (preferably human), also referred to as cell-free fetal DNA (cffDNA) (Barrett et al., PloS One (2011 ), 6: e25202) and cell-free maternal DNA (cfmDNA), respectively.
  • the amount of cffDNA in relation to the total amount of the maternal cfDNA in the same sample is also referred to as fetal fraction ( ).
  • Such cffDNA may be present as segments or fragments and sequenced by Massive Parallel Sequencing (MPS) methods known in the art or other suitable sequencing methods (e.g., NGS, second or third generation sequencing) and the resulting reads may be aligned to a human genome and counted, e.g., by methods known in the art and described and exemplified herein.
  • MPS Massive Parallel Sequencing
  • NGS second or third generation sequencing
  • a chromosome e.g., 21 , 18, 13, 1 , 5, or X
  • parts thereof e.g., 22, 12p, 9p, 18p
  • an aneuploidy e.g., tetrasomy, trisomy or monosomy, respectively
  • an aneuploidy e.g., tetrasomy, trisomy or monosomy, respectively
  • chromosome-specific reads exceeds the threshold that represents a normal (euploid or disomic) chromosome status
  • the result is reported as positive for trisomy for that chromosome (Norwitz et al., Rev Obstet Gynecol (2013), 6(2): 48-62).
  • the same holds true vice versa for falling below such threshold which is then reported as positive for monosomy for that chromosome.
  • PCR polymerase chain reaction
  • NASBA nucleic acid sequence based amplification
  • Preferred amplification methods involve PCR.
  • the amplification uses at least one fetal sequence specific oligonucleotide primer.
  • the nucleic acid may be detected by means of a sequence specific probe.
  • the LoD may be determined by methods known on the art and also described herein (e.g., Fiorentino et al., Prenat. Diagn. 36, 304-311 (2016). URL http://dx.doi.org/10.1002/pd.4780).
  • the LoD may for example be determined experimentally as known in the art and as described herein.
  • the LoD may be different for each respective chromosome for which an aneuploidy (e.g., monosomy or trisomy) shall be determined in accordance with the methods of the present invention as described and provided herein.
  • the set of Rano call samples comprises all samples that have fetal fraction f below the LoD.
  • Such favorno call ‘Samples are then usually disregarded and discarded. That is, in such conventional NIPT methods a sample with fetal fraction below LoD is considered as contestno call 3 ⁇ 4nd thus disregarded and discarded, regardless of its read representation of a chromosome t. Improved methods for determining the suitability of a given sample for testing are described in EP17203198.
  • Circulating cell free DNA comprises (fragments of) nucleic acid (DNA, RNA; preferably DNA) molecules of different length, which are not organized in cell nuclei and not separated by membrane from outer medium, but which are localized in liquid medium within a given subject (preferably mammal, more preferably human) such as urine, plasma, or serum (preferably plasma).
  • cfDNA can be found in samples from a pregnant subject (preferably mammal, preferably human), e.g., from maternal plasma samples.
  • the skilled person is aware of methods to measure and count nucleic acid (particularly DNA) molecules in (plasma) samples, particularly reads from cfDNAas described herein.
  • MPS massive parallel sequencing
  • methods as known in the art and also described and exemplified herein allow fast counting of myriads of nucleic acid (particularly DNA) molecules, e.g. reads as described herein (Lo, Open Biol. (2012), 2(6): doi 10.1098/rsob.120086).
  • Further methods for detecting cfDNA, cffDNA or cfmDNA are known in the art and described, e.g., in Nigam et al., JIMSA (2012), 25: 119-200.
  • Circulating cell free fetal DNA comprises cfDNA fragments of fetal origin in a given sample of the pregnant mother of the fetus (preferably mammal, more preferably human), preferably cfDNA fragments of fetal origin in the plasma of a pregnant human.
  • the skilled person is aware of methods to measure and count nucleic acid (particularly DNA) molecules in (plasma) samples, particularly reads from cffDNA as described herein.
  • MPS massive parallel sequencing
  • Circulating cell free maternal DNA cfmDNA
  • maternal cfDNA comprises cfDNA fragments of maternal origin in a given sample of the pregnant mother, preferably cfDNA fragments of maternal origin in the plasma of a pregnant human.
  • the skilled person is aware of methods to measure and count nucleic acid (particularly DNA) molecules in (plasma) samples, particularly reads of cfDNA, cfmDNA and cffDNA as described herein.
  • MPS massive parallel sequencing
  • Fetal fraction (f) means the amount of cffDNA in relation to the total amount of the maternal cfDNA in the same sample (expressed as percentage).
  • the fetal fraction also known as fractional fetal DNA concentration
  • the fetal fraction can be measured by means and methods known in the art.
  • the fetal fraction may be measured, e.g., in placental samples, or maternal blood plasma samples from pregnant women, where the specific signal originating from Y chromosome may be used to deduce the fetal DNA fractions in pregnancies carrying male fetuses (Chiu at al., BMJ (201 1 ), 342: c7401 ; Hudecova et al., PLoS One (2014), 9: e88484; also known as ffY method”).
  • NIPS Read -counting approach to NIPS as used herein refers to the read counting-based NIPS as proposed independently by (Fan et al., doi 10.1073/pnas.0808319105, PNAS (2008)) and (Chiu et al., PNAS 105(51 ), 20458-20463 (2008)).
  • steps in NIPS may be comprise: first, a sample is subjected to WGS. The sequenced reads are then aligned to a reference human genome. Afterwards, the low-quality and duplicate reads are filtered out.
  • Aligned reads are binned and normalized. Corrections, including the GC correction, may be applied.
  • the number of normalized reads in a chromosome, or a subset of the chromosome is known as the chromosome representation, or the read representation of the chromosome.
  • the chromosome representation is one of the two quantities that characterize a sample in NIPS. The other one is fetal fraction. Fetal fraction is the relative amount of fetal (more precisely, placental) cfDNA to the total (maternal and placental) cfDNA.
  • the NIPS may further be calibrated by generating mono-, di-, tri-, and/or tetrasomy bands as described and provided herein.
  • Massive Parallel Sequencing means simultaneous, massive, parallel sequencing of an extremely large amount of DNA molecules. It is also referred to in the art and herein as next generation sequencing” or NGS”, or as second generation sequencing” (for some sequencing methods also third generation sequencing’).
  • the sequencing data i.e., reads
  • the reference e.g., human genome
  • bioinformatic tools as known in the art and also described and exemplified herein (e.g., Bowtie2 as described in Langmead et al., Nat Methods (2012), 9: 357-359). Unmapped, duplicate, low quality reads may be filtered out by suitable tools as known in the art.
  • the reads mapped (i.e., aligned) to a particular chromosome e.g., 21 , 18, 13, 5, 1 , X
  • parts thereof e.g., 22, 12p, 9p, 18p
  • the reads count may then be compared to the number of reads in the chromosome of one or more samples which are euploid in the chromosome (Norwitz et al., Rev Obstet Gynecol (2013), 6(2): 48-62).
  • MPS methods comprise inter alia Polony sequencing, 454 (pyro)sequencing, llumina dye sequencing, SOLiD sequencing, DNA nanoball sequencing, SMRT sequencing, Nanopore sequencing, and others (cf, e.g., WO 9844151 ; Duncan et al., Diagn Mol Pathol (2017), doi.
  • Read as used herein means a sequence of segment or fragment of DNA, for example which may be derived from a particular chromosome (e.g., 21 , 18, 13, 5, 1 , or X) or parts thereof (e.g., 22, 12p, 9p, 18p), obtained by sequencing, e.g. by MPS methods; cf. Bayindir et al., Eur. J. Hum. Genet. 23, 1286- 1293 (2015); Porreco et al., Am. J. Obstet. Gynecol. 211 , 365- e1 (2014); Thung et al., Expert. Rev. Mol. Diagn. 15, 11 1 - 124 (2015); US2016224724; EP2981921.
  • MPS methods usually such reads of cfDNA, cfmDNA and cffDNA are obtained by MPS methods as known in the art and described herein.
  • Read representation of a chromosome (t)” or dhromosome representation (t)” means the number of reads mapped to a particular chromosome or subregions thereof (also referred to herein as (‘chromosomal) read counts’) and optionally filtered, preprocessed and normalized. Normalization can be done as described herein and further shown, e.g., in Bayindir et al., Bur. J. Hum. Genet. 23, 1286- 1293 (2015); Porreco et al., Am. J. Obstet. Gynecol. 21 1 , 365- e1 (2014); Thung et al., Expert. Rev. Mol. Diagn.
  • LoD Limit of Detection
  • each chromosome (e.g., 13, 18, 21 , X, 1 , 5) or parts thereof (e.g., 22, 12p, 9p, 18p) has its own LoD for monosomy, trisomy.
  • the LoD for trisomy of chromosome 21 may be about 3 to 4.5%.
  • the LoD for trisomy of chromosome 18 may be about 3.5 to 6.5%.
  • the LoD for trisomy of chromosome 13 may be about 3.2 to 5.8%.
  • the LoD for monosomy of chromosome 21 or 18 may be about 3 to 4%.
  • the LoD for monosomy of chromosome 13 may be about 3.5 to 4.5%.
  • for detection of trisomy can be decreased by decreasing the range of euploid read representation of a chromosomes
  • the LoD for determining particular chromosome aneuploidies may also be dependent on the method for normalization of the reads as shown and described herein.
  • No call’ sample as used herein means a sample for which the degree of monosomic, trisomic, tetrasomic mosaicism cannot be determined with the sufficiently small uncertainty, where the uncertainty is measured by the width of the monosomic, trisomic, tetrasomic interval.
  • the set of all no call samples for a given maximal tolerable width w’ of the trisomic mosaicisim interval is called the blind spot for determination of the degree of trisomic mosaicism with uncertainty at most w’.
  • the set of all no call samples for a given maximal tolerable width v’ of the monosomic mosaicisim interval is called the blind spot for determination of the degree of monosomic mosaicism with uncertainty at most v’.
  • the set of all no call samples for a given maximal tolerable width u’ of the tetrasomic mosaicisim interval is called the blind spot for determination of the degree of tetrasomic mosaicism with uncertainty at most u’.
  • aneuploidy means the presence of an abnormal number of (portions of) chromosomes in a cell and comprises any kind of deviation from the diploid chromosome set where two homologous copies of each autosome (e.g., for human beings, chromosomes 1 to 22) and two allosomes (sex chromosomes; e.g., for mammals such as humans two copies of the X chromosome for females and one copy of the X and one copy of the Y chromosome for males) are present in a cell of a given subject (e.g., fetus; human fetus), where one copy of each chromosome is received from the mother and one from the father, respectively (for sex chromosomes in mammals like humans, one X is received from the mother, while the other X or the Y, respectively, is received from the father of the individuum).
  • two homologous copies of each autosome e.g., for human beings,
  • cineuploidy may also be used herein interchangeably with the terms Numerical chromosomal abnormality”, Numerical chromosomal aberration” or humeri cal chromosomal disorder” as known in the art.
  • Aneuploidy as used herein also comprises abnormal number of individual chromosomes, e.g. only one copy of a specific chromosome (i.e. a monosomy; e.g., monosomy of chromosome 13, 18, 21 or X), or three copies of a specific chromosome (i.e.
  • a trisomy e.g., trisomy of chromosome 13, 18, 21 or X
  • four copies of a chromosome or parts thereof i.e. a tetrasomy; e.g., tetrasomy of parts of chromosome 22 (cat eye syndrome), tetrasomy of the short arm (p) of chromosome 12 (12p; Pallister-Killian syndrome), tetrasomy 9p, or tetrasomy 18p; or tetrasomy of gonosomes like XXXX or XXYY).
  • the term aneuploidy also comprises trisomy”, tetrasomy” or monosomy”.
  • Trisomy as used herein means that for at least one chromosome, there are three copies of a chromosome present in a cell within the set of chromosomes of a given subject (e.g., fetal set of chromosomes).
  • trisomy may comprise a trisomy for one or more of chromosomes 21 ( tfisomy 21”), 18, 13, 9, 8, 22 or X, where three copies of at least one (preferably only one) of said chromosomes are present in at least a subset of cells or all cells of the subject (fetus, e.g., human fetus), or a XXY or XYY trisomy where three sex chromosomes are present.
  • trisomy means three copies of chromosome 21 ( Down syndrome”, trisomy 21”), 18 ( Edwards syndrome” , trisomy 18’), 13 ( Patau syndrome”, trisomy 13’), 8 ( Warkany syndrome 2” , trisomy 8’), X ( Triple X syndrome” , trisomy X’), or three sex chromosomes other than XXX, namely XYY ( Jacobs syndrome’) or XXY ( Klinefelter syndrome’), preferably trisomy 21 , 18, 13 or X, or three sex chromosomes other than XXX, namely XYY ( Jacobs syndrome’) or XXY ( Klinefelter syndrome’).
  • the fetal trisomy status is trisomy 13, 18, 21 or
  • the trisomic mosaicism may concern any autosome or chromosome X.
  • the trisomic mosaicsm concerns any one of chromosomes 13, 18, 21 or X.
  • Monosomy as used herein means that for at least one chromosome, there is only one copy of a chromosome present in a cell within the set of chromosomes of a given subject (e.g., fetal set of chromosomes). As almost all full monosomies are lethal (except, e.g., mammal (human) monosomy for X where also no Y is present (XO; Turner syndrome’), the term monosomy” as used herein also comprises partial monosomy” such as, e.g.
  • chromosome 1 for mammals, humans
  • chromosome 5 Cri-du-chat syndrome
  • structural chromosomal aberration or structural chromosomal disorder in at least a subset of cells or all cells of the subject (fetus, e.g., human fetus).
  • monosomy as used herein also comprises partial monosomy” where only one copy of a particular chromosome (autosome or sex chromosome) is present or part or a segment of a particular chromosome is missing in at least a subset of cells or all cells of the subject (fetus, e.g., human fetus).
  • monosomy may comprise a monosomy for example (particularly in case the subject is mammal, e.g., human) for one or more of chromosomes 21 , 18, 13, 1 or 5 or parts thereof, where only one copy of said chromosome is present or part of one of said chromosomes is missing (also partial monosomy’) in a cell or all cells of the subject (fetus, e.g., human fetus), or a XO monosomy where only one sex chromosome (X) is present.
  • a monosomy for example (particularly in case the subject is mammal, e.g., human) for one or more of chromosomes 21 , 18, 13, 1 or 5 or parts thereof, where only one copy of said chromosome is present or part of one of said chromosomes is missing (also partial monosomy’) in a cell or all cells of the subject (fetus, e.g., human fetus), or
  • rVionosomy means only one copy of chromosome 21 , 18, 13, or missing part or segment of chromosome 1 ( fp36 deletion syndrome”, (partial) monosomy 1’) or chromosome 5 ( Cri-du-chat syndrome”, (partial) monosomy 5’), or only one copy of X (and no Y) ( Turner syndrome”, monosomy X”, XO).
  • the fetal monosomy status is (partial) monosomy 21 , 18, 13, 5 or X, or particularly monosomy 21 , 18, or 13 (or X).
  • the monosomic mosaicism may concern any autosome or chromosome X.
  • the monosomic mosaicsm concerns any one of chromosomes 21 , 18, 13, 5 orX, or particularly monosomy 21 , 18, or 13 (or X).
  • Tetrasomy as used herein means that for at least one chromosome or parts thereof, there are four copies present in a cell within the set of chromosomes of a given subject (e.g., fetal set of chromosomes). That is, four copies of a chromosome or parts thereof may be present in at least a subset of the cells of a given subject (i.e.
  • tetrasomy e.g., tetrasomy of parts of chromosome 22 (cat eye syndrome), tetrasomy of the short arm (p) of chromosome 12 (12p; Pallister-Killian syndrome), tetrasomy 9p, or tetrasomy 18p; or tetrasomy of gonosomes like XXXX or XXYY).
  • tetrasomy also comprises partial tetrasomy” for only parts of a given chromosome.
  • the tetrasomic mosaicism may concern any autosome or chromosome X.
  • the tetrasomic mosaicsm concerns any one of parts of chromosome 22 (cat eye syndrome), tetrasomy of the short arm (p) of chromosome 12 (12p; Pallister-Killian syndrome), tetrasomy 9p, or tetrasomy 18p; or tetrasomy of gonosomes like XXXX or XXYY.
  • a sample may not qualify for the determination of the degree of monosomic, trisomic, and tetrasomic mosaicism, if at least one of the widths of the monosomic, trisomic or tetrasomic mosaicism intervals, respectively, is greater than or equal to the corresponding maximal tolerable width of the respective mosaicism interval.
  • Chromosomal mosaicism is the presence of two or more distinct cell lines in an individual (Taylor et al. Human Reproduction Update 20(4), 571 -581 (2014)). There are two forms of the mosaicism: general mosaicism and confined mosaicism.
  • General mosaicism affects both fetus and placenta. When the mosaicism affects only some organs (e.g., brain, placenta, gonads), it is referred to as the confined mosaicism. Thus, there is the confined placental mosaicism, which affects solely placenta.
  • the percentage of trisomic cells need not be the same at all the affected regions of placenta (Canick et al., Prenatal Diagnosis 33(7), 667-674 (2013)).
  • the percentage of trisomic cells may be different in different regions affected by the placental mosaicism.
  • Each region, affected by some degree of mosaicism contributes the cfDNA into the maternal blood.
  • the screening result refers to the aggregated or averaged percentage of mosaicism. In other words, NIPS cannot measure the percentage of mosaicism at a particular region of placenta. NIPS can estimate the averaged percentage of mosaicism, i.e., the degree of mosaicism.
  • mosaicism refers to the presence of differing population of cells with different genotypes which has developed from a single fertilized cell, particularly to different ploidy status between different cells.
  • a part of the cells of a given subject may contain a monosomic, trisomic or tetrasomic status for a given chromosome, while another part of the subject’s cells is euploid (diploid) for that given chromosome.
  • rthosaicism also refers to the state where a subset or even all (i.e.
  • the cells of a given subject have a genotype, particularly ploidy status, deviating from the euploid (diploid) status.
  • a genotype particularly ploidy status
  • ploidy status deviating from the euploid (diploid) status.
  • the monosomic mosaicism interval, trisomic mosaicism interval, and tetrasomic mosaicism interval quantify the uncertainty of determining the degree of monosomic, trisomic, tetrasomic mosaicism; respectively.
  • the intervals may generally serve to define the interval-based estimate of the degree of monosomic, trisomic, tetrasomic mosaicism; respectively.
  • maternal trisomic, monosomic and tetrasomic respectively, degree is 0 and the fetal (e.g., placental) mosaicism is determined.
  • chromosome representation where is the read representation of the chromosome of the sample (also referred to herein as chromosome representation),
  • the chromosome representation of a mosaic sample with fetal fraction f is related to the chromosome representation of an euploid sample by the formula
  • the term in the square brackets (i.e. [( 1 - ) ⁇ + 1.5 ⁇ ⁇ ]) is composed of the euploid contribution ( 1 - ) ⁇ and the trisomic contribution 1.5 ⁇ ⁇ .
  • the degree of trisomic mosaicism would be 100 % (i.e. all cells exhibit a trisomic state for a given chromosome). Accordingly, to exclude 100% mosaicism, in one embodiment of the present invention, in formula (A), Y is not 1.
  • every an euploid mother with an euploid placenta has the same chromosome representation .
  • the degree g of trisomic placental mosaicism can be determined with absolute precision by solving formula A for , which leads to formula (B):
  • the chromosome representation of euploid mother with euploid placenta varies in a range of values [ ] .
  • the main source of the variability is the technical variability of the
  • the present invention also relates to a method for determining whether or not a sample (e.g., pregnant/maternal; placental; fetal subject) characterized by its fetal fraction , the read representation of the chromosome and the width of trisomic mosaicism interval qualifies for determination of the degree of trisomic mosaicism with the uncertainty at most ', comprising determining whether ⁇
  • the present invention also provides an embodiment where said method for determining whether or not a sample qualifies for determination of the degree of trisomic mosaicism with the uncertainty at most ' is equivalent to determining whether where
  • w’ is set to 1 , then a sample may be considered to fail the Quality Control criterion if its trisomic mosaicism interval is [0,1] For this embodiment of the present invention, for such a sample the aneuploidy status is considered not to be determinable. In a specific embodiment of the present invention, w’ is not 1.
  • the read representation of a chromosome may also be replaced by the z-scone wherein m is an estimate of the location of the distribution of the read representation of the chromosome in a population that is euploid in the chromosome, is an estimate of the scale of the distribution of the read representation of the chromosome in a population that is euploid in the chromosome, comprising determining the uncertainty of the degree of trisomic mosaicism in a sample by the trisomic mosaicism interval [ ] with the width
  • samples are qualified for determining the fetal trisomy status of a chromosome according to the following determination:
  • any one of (a) to (c) is not fulfilled.
  • samples are qualified for determining the fetal trisomy status of a chromosome according to the following determination:
  • the present invention also relates to a method for determining the uncertainty of the degree of monosomic mosaicism in a sample by the monosomic mosaicism interval [ ] with the width
  • p 1
  • the degree of monosomic mosaicism would be 100 % (i.e. all cells exhibit a monosomic state for a given chromosome). Accordingly, to exclude 100% mosaicism, in one embodiment of the present invention, in formula (C), p is not 1.
  • the present invention further relates to a method for determining whether or not a sample characterized by its fetal fraction , the read representation of the chromosome and the width of monosomic mosaicism interval as defined above qualifies for determination of the degree of monosomic mosaicism with the uncertainty at most comprising determining whether ⁇ ', wherein if 3 ' said sample is not qualified for determining the degree of monosomic mosaicism with uncertainty at most ', or
  • said method for determining whether or not a sample qualifies for determination of the degree of monosomic mosaicism with the uncertainty at most ' may be equivalent to determining whether
  • fetal fraction of the sample is the minimal value of the read representation of the chromosome in a set of euploid samples
  • v’ is set to 1 , then a sample may be considered to fail the Quality Control criterion if its trisomic mosaicism interval is [0,1]. For this embodiment of the present invention, for such a sample the aneuploidy status is considered not to be determinable. In a specific embodiment of the present invention, v’ is not 1.
  • samples are qualified for determining the fetal monosomy status of a chromosome according to the following determination:
  • samples are qualified for determining the fetal monosomy status of a chromosome according to the following determination:
  • any one of (a) to (c) is not fulfilled.
  • the present invention further relates to a method for determining the uncertainty of the degree of tetrasomic mosaicism relative to trisomy in a sample from euploid mother by the tetrasomic mosaicism interval [ . ] with the width
  • the degree of the tetrasomic mosaicism relative to euploidy, the chromosome representation of an euploid mother with the mosaic placenta, the chromosome representation t E of euploid mother with euploid placenta, and fetal fraction are related together by
  • formula (D) permits to find the bounds [ ] of the tetrasomic mosaicism interval to be
  • the degree of tetrasomic mosaicism would be 100 % (i.e. all cells exhibit a tetrasomic state for a given chromosome). Accordingly, to exclude 100% mosaicism, in one embodiment of the present invention, in formula (D), w is not 1.
  • the degree of tetrasomic mosaicism may also be expressed in relation to trisomy band. For instance, a sample with the degree of tetrasomic mosaicism 0.6 has the 60% of placental cells with four copies of a given chromosome, and 40% of placental cells with two copies of that given chromosome.
  • the tetrasomic mosaicism interval [ ] expressed relative to trisomy may be given as:
  • the present invention further relates to a method for determining whether or not a sample from euploid mother characterized by its fetal fraction , the read representation of the chromosome and the width of tetrasomic mosaicism interval as defined above qualifies for determination of the degree of tetrasomic mosaicism with the uncertainty at most comprising determining whether ⁇ ',
  • said method for determining whether or not a sample from euploid mother qualifies for determination of the degree of tetrasomic mosaicism with the uncertainty at most ' may be equivalent to determining whether
  • the read representation of a chromosome may be replaced by the z-score
  • m is an estimate of the location of the distribution of the read representation of the chromosome in a population that is euploid in the chromosome, is an estimate of the scale of the distribution of the read representation of the chromosome in a population that is euploid in the chromosome, comprising determining the uncertainty of the degree of tetrasomic mosaicism in a sample from euploid mother by the tetrasomic mosaicism interval [ ] with the width
  • a sample may not qualify for the determination of the degree of monosomic, trisomic, and tetrasomic mosaicism, if at least one of the widths of the monosomic, trisomic or tetrasomic mosaicism intervals, respectively, is greater than or equal to the corresponding maximal tolerable width of the respective mosaicism interval.
  • Chromosomal mosaicism is the presence of two or more distinct cell lines in an individual (Taylor et al. Human Reproduction Update 20(4), 571 -581 (2014)). There are two forms of the mosaicism: general mosaicism and confined mosaicism.
  • General mosaicism affects both fetus and placenta. When the mosaicism affects only some organs (e.g., brain, placenta, gonads), it is referred to as the confined mosaicism. Thus, there is the confined placental mosaicism, which affects solely placenta.
  • the percentage of trisomic cells need not be the same at all the affected regions of placenta (Canick et al., Prenatal Diagnosis 33(7), 667-674 (2013)).
  • the percentage of trisomic cells may be different in different regions affected by the placental mosaicism.
  • Each region, affected by some degree of mosaicism contributes the cfDNA into the maternal blood.
  • the screening result refers to the aggregated or averaged percentage of mosaicism. In other words, NIPS cannot measure the percentage of mosaicism at a particular region of placenta. NIPS can estimate the averaged percentage of mosaicism, i.e., the degree of mosaicism.
  • mosaicism refers to the presence of differing population of cells with different genotypes which has developed from a single fertilized cell, particularly to different ploidy status between different cells.
  • a part of the cells of a given subject may contain a monosomic, trisomic or tetrasomic status for a given chromosome, while another part of the subject’s cells is euploid (diploid) for that given chromosome.
  • mosaicism also refers to the state where a subset or even all (i.e.
  • the cells of a given subject have a genotype, particularly ploidy status, deviating from the euploid (diploid) status.
  • a genotype particularly ploidy status
  • ploidy status deviating from the euploid (diploid) status.
  • the monosomic mosaicism interval, trisomic mosaicism interval, and tetrasomic mosaicism interval quantify the uncertainty of determining the degree of monosomic, trisomic, tetrasomic mosaicism; respectively.
  • the intervals may generally serve to define the interval-based estimate of the degree of monosomic, trisomic, tetrasomic mosaicism; respectively.
  • interval -based estimate of the degree of trisomic mosaicism may be defined as
  • the interval-based estimate of the degree of monosomic mosaicism may be defined as
  • the interval-based estimate of the degree of tetrasomic mosaicism relative to trisomy may be defined as
  • the degree of mosaicism (mononosomic, trisomic, tetrasomic) can be determined up to the interval of (mononosomic, trisomic, tetrasomic) mosaicism.
  • the width of the (monosomic, trisomic, tetrasomic) interval quantifies the uncertainty of the determination of the degree of the (monosomic, trisomic, tetrasomic) mosaicism.
  • the width of the trisomic mosaicism interval may then be - .
  • a sample passes the QC criterion if £ ', and £ and £ '
  • Non-lnvasive Prenatal Screening (NIPS) system may be applied for measuring and quantifying the degree of mosaicism (e.g., for tri-, mono-, or tetrasomy) of a given sample (e.g., pregnant/maternal; placental; fetal subject).
  • a given sample e.g., pregnant/maternal; placental; fetal subject.
  • the present invention further relates to a method for calibrating a Non-lnvasive Prenatal Screening (NIPS) system, comprising
  • the monosomy band is as follows: all pairs (f, t) of fetal fraction f and the read representation of the chromosome t such that
  • the disomy band is as follows: all pairs (f, t) of fetal fraction f and read representation of the chromosome t such that
  • the trisomy band is as follows: all pairs (f, t) of fetal fraction f and the read representation of the chromosome t such that
  • the tetrasomy band is as follows: all pairs (f, t) of fetal fraction f and the read representation of the chromosome t such that
  • step (b) determining whether or not on the basis of processed sequencing data obtained from a plurality of blood samples from pregnant subjects comprising circulating fetal DNA, wherein the fetal karyotype corresponding to each of the samples is known, said samples with the monosomic karyotype fall with the monosomy band, said samples with the trisomic karyotype fall with the trisomy band, and said samples with the tetrasomic karyotype fall with the tetrasomy band as generated in step (a),
  • the NIPS system is not well-calibrated.
  • the present invention further relates to the items as listed below in context with the methods described and provided herein:
  • a method for determining whether or not a sample characterized by its fetal fraction , the read representation of the chromosome and the width of tetrasomic mosaicism interval as defined in item 13 qualifies for determination of the degree of tetrasomic mosaicism with the uncertainty at most comprising determining whether ⁇ ',
  • a method for calibrating a Non-lnvasive Prenatal Screening (NIPS) system comprising
  • the monosomy band is as follows: all pairs (f, t) of fetal fraction f and the read representation of the chromosome t such that
  • the disomy band is as follows: all pairs (f, t) of fetal fraction f and read representation of the chromosome t such that
  • the trisomy band is as follows: all pairs (f, t) of fetal fraction f and the read representation of the chromosome t such that
  • the tetrasomy band is as follows: all pairs (f, t) of fetal fraction f and the read representation of the chromosome t such that
  • the NIPS system is not well-calibrated.
  • the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention. Also, the term at least” preceding a particular amount or number also comprises exactly this amount or number, i.e. it may also serve as specification of a particular amount or number.
  • dbnsisting of excludes any element, step, or ingredient not specified in the claim element.
  • Consisting essentially of' does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.
  • Figure 1 The blind spot for determination of the degree of trisomic mosaicism with uncertainty at most 20% is depicted as the quadrangle with the 135 degree line shading in the (f, t) plane.
  • the sample according to Example 1 is depicted as the triangle. It lays inside the blind spot, which implies that the degree of trisomic mosaicism for this particular sample cannot be determined with the uncertainty at most 20%; rather, the width w of the trisomic mosaicism interval for this sample is greater than 20%.
  • the sample according to Example 2 is depicted as the square.
  • the sample lays outside the blind spot, implying that the width of the trisomic mosaicism interval is smaller than 20% and hence the sample passes the QC criterion (i.e. it is qualified for determination of the degree of trisomic mosaicism so that the degree of trisomic mosaicism according to the present invention), estimated by the interval-based estimate can be reported to the patient, together with the trisomic mosaicism interval.
  • Figure 2 The values of the read representation t of chromosome 21 and fetal fraction f for
  • Figure 4 Trisomic and monosomic blindspots
  • the blind spot for monosomic and trisomic mosaicism, when the maximal tolerable uncertainty v’ for monosomic mosaicism, w’ for trisomic mosaicism are set to 0.2, 0.4, 0.8 and 1 ; respectively.
  • the size (area) of the blind spot decreases, as the maximal tolerable uncertainty increases.
  • the disomy (euploidy) band is the fundamental band, from which the monosomy, trisomy and tetrasomy band are derived.
  • the disomy band is defined as all the pairs (f, t) of fetal fraction f and the chromosome representation t for which t is in
  • the minimal and maximal values of the chromosome representation is obtainable from a cohort of samples with confirmed euploid karyotype.
  • FIG. 7 Maternal trisomic mosaicism of known degree and placental trisomic mosaicism (T m T g )
  • a sample with z-score 15.6 and fetal fraction 0.1 could be considered tetrasomic mosaic, if the mother was assumed euploid; or it could have 50% to 100% trisomic mosaicism if the mother was known to have 7% trisomic mosaicism.
  • the dashed green lines delineate the trisomy band.
  • the band is associated with the conventional [- 3,3] disomy band (not shown).
  • the blue solid lines delineate the band. Note that 0.75 is the average of the lower (0.5) and upper (1.0) bounds on g.
  • FIG. 8 Maternal trisomic mosaicism of known degree and placental trisomic mosaicism (T m T g )
  • a sample with z-score 0 and fetal fraction 10% (the red spot) could be considered to indicate the euploid placenta if the mother was known to be euploid, or it could indicate 62% (26% to 99%) monosomic placental mosaicism, if the mother was known to have 7% trisomic mosaicism.
  • FIG. 10 Maternal monosomic mosaicism of known degree and placental trisomic mosaicism (M m T g )
  • a sample with z-score - 6.6 on chromosome X and fetal fraction 10% suggest male gender - since the sample lays in the bby band’ (dashed green lines), under the assumption of maternal healthy karyotype. If the 10% maternal X0 mosaicism is assumed, then the sample has 0% to 36% XXX mosaicism; i.e., it is compatible with XX.
  • the blue solid line represents the z-score of mosaic mother and corresponds to the lowest z-score among XX samples; i.e. - 2.7.
  • Step 1 The Whole Genome Massive Parallel Sequencing (MPS), bioinformatic processing, reads mapping, filtering, binning, read representation of a chromosome cfDNA from a blood sample from a pregnant woman was sequenced by MPS.
  • the resulting reads were mapped to the human genome hg19 by the algorithm bowtie2. Duplicate and low-quality reads were afterwards filtered out. Then the reads were binned into 50 kilo base pair bins.
  • the total number of reads in a subset of bins was normalized by the total read counts in all autosomes.
  • the subset of bins was selected by an in house method. Selection of the subset has not assumed information about fetal fraction, thus avoiding circularity. Moreover, the subset selection was cross-validated.
  • Step 2 Determination of the minimal and maximal values of the read representation of the chromosome 21 in euploid pregnancies.
  • the minimal value of the read representation of the chromosome 21 in the same subset of bins as used in Step 1 , was equal to 0.0078291.
  • the maximal value was 0.0079678.
  • Fetal fraction for the pregnancy was estimated by the method seqFFY to be 0.15 (i.e., 15%).
  • the seqFFY method for estimating fetal fraction has been performed as follows:
  • seqFF seqFF
  • the calibration curve can be estimated by any of the regression fitting methods; the cross-validated smoothing spline (R Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria (2015). URL https://www.R-project.org/) was used to obtain the calibration curve.
  • Step 4) Determination of the trisomic mosaicism interval and the degree of the trisomic mosaicism
  • the same conclusion as that reached on Step 5) could be obtained by checking formulas (1 ), (2) and (3):
  • the width w of the trisomic mosaicism interval was 0.193 (i.e., 19.3%).
  • the interval-based of the degree of trisomic mosaicism was 0.117 (i.e., 1 1.7%). Since the width w was smaller than the preselected maximal tolerable width w’ of the trisomic mosaicism interval, the sample qualified for determination of the degree of the trisomic mosaicism.
  • t was greater than and hence the monosomic mosaicism interval (and the interval-based estimate of the degree of monosomic mosaicism) was not defined for the sample.
  • the tetrasomic interval of mosaicism did not exist for this sample, either.
  • the sample qualified for determination of the degree of trisomic mosaicism and the interval -based estimate (11.7%) of the degree of trisomic mosaicism together with the interval itself ([0.020, 0.213]) could be reported to the patient.
  • Such a sample would be considered to have a tetrasomic placental mosaicism if the mother was euploid.
  • this sample would have 50% to 100% trisomic placental mosaicism.
  • the trisomic mosaicism could erroneously be considered tetrasomic, if the mother was assumed euploid.
  • a sample with 10% fetal fraction and z-score 13.2, and the range of z-score for euploid samples is [- 3,3]. If the mother is known to be 10% mosaic on the chromosome, then the placental trisomic mosaicism can be estimates to be in [0,0.58], i.e., the sample is compatible with placental euploidy. However, if the mother was assumed euploid, the placenta would be compatible with trisomy, as the degree of trisomic placental mosaicism would be in the interval [0.9,1]
  • Samples with two copies of a chromosome have a z-score in [- 27,2.7], as is the case for a subset of chromosome X, that is used for gender determination. Then, assuming that mother is euploid (i.e., has two copies of the chromosome), the sample would be considered euploid as well.
  • XXX trisomic mosaicism
  • this sample would have 26% to 99% (average, 62%) monosomic placental mosaicism; hence, it permits male gender of the placenta.
  • XXX trisomic mosaicism
  • sample with fetal fraction 10% and the z-score - 2 Provided is a sample with fetal fraction 10% and the z-score - 2.
  • Samples with two copies of a chromosome have a z-score in [- 3,3], as is the case for (a subset of) chromosome 21. Then, assuming that mother is euploid (i.e., has two copies of the chromosome), the sample would be considered euploid as well.
  • this sample would have 80% to 100% (average, 90%) monosomic placental mosaicism.
  • an euploid placenta from an euploid mother is indistinguishable from a monosomic placenta from a mother with 10% T21 mosaic.

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Abstract

La présente invention concerne le dépistage prénatal non invasif (NIPS). En particulier, la présente invention concerne des procédés pour déterminer l'incertitude du niveau de mosaïcisme placentaire dans un échantillon prélevé chez une femme enceinte. Selon la présente invention, la largeur associée des intervalles de mosaïcisme monosomiques, trisomiques et tétrasomiques est utilisée pour déterminer le niveau de mosaïcisme monosomique, trisomique et tétrasomique (placentaire). La présente invention concerne en outre des procédés pour déterminer si un échantillon remplit ou non les conditions requises pour déterminer le niveau de mosaïcisme placentaire dans le cadre de l'approche de dénombrement de séquences de fragments (read-counting) en NIPS.
PCT/EP2020/054287 2019-02-20 2020-02-19 Procédé pour déterminer l'incertitude du niveau de mosaïcisme placentaire d'un échantillon dans un dépistage prénatal non invasif WO2020169635A1 (fr)

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