(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
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`(10) International Publication Number
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`g
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`(43) International Publication Date
`WO 2014/068075 A1
`WIPOI PCT
`3 May 2014 (08.05.2014)
`
`
`(51)
`
`International Patent Classification:
`CIZQ 1/68 (2006.01)
`
`(21)
`
`International Application Number:
`
`PCT/EP2013/072848
`
`(22)
`
`International Filing Date:
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`(25)
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`Filing Language:
`
`(26)
`
`Publication Language:
`
`31 October 2013 (31.10.2013)
`
`English
`
`English
`
`(30)
`
`(71)
`
`(72)
`
`Priority Data:
`121908446
`
`31 October 2012 (31.10.2012)
`
`EP
`
`Applicant: GENESUPPORT SA [CH/CH]; Route de Ber-
`tigny 36, CH-1700 Fribourg (CH).
`
`Inventors: ESTERAS, Nlagne; 19, avenue Bois-de-la-
`Chapelle, CII-1213 Onex (CII). DELUEN SAGNE. Cé-
`cile; 50, ehemin du Dessy, F-01630 Sergy (FR). VIN-
`CENT, Nadine;
`1, rue du Pre Leavard, F-01200 In-
`jouX-Genissiat
`(FR). CONRAD, Bernard;
`Jardins du
`Salesianum 11, CH-1700 Fribourg (CH).
`
`(74)
`
`Agents: ERNEST GUTMANN - YVES PLASSERAUD
`SAS et al.; 88, Bd des Belges, F-69452 LYON Cedex 06
`(FR).
`
`(81)
`
`Designated States (unless otherwise indicated, for every
`kind ofnational protection available): AE, AG, AL, AM,
`A0, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, Fl, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ,
`OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA,
`SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM,
`TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM,
`ZW.
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RWY, SD, SL, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GVV,
`KM, ML, MR, NE, SN, TD, TG).
`Published:
`
`with international search report (Art. 21(3))
`
`(54) Title: NON-INVASIVE METHOD FOR DETECTING A FETAL CHROMOSOMAL ANEUPLOIDY
`
`(57) Abstract: The invention relates to a method for obtaining a set of reference samples and/or a set of reference parameters for the
`diagnosis of fetal ancuploidy front a maternal biological sample, containing cell-free DNA, said method comprising: - extracting
`cell-free DNA from a set of biological samples obtained from euploid pregnant women carrying a euploid fetus; - after the extraction
`step, analyzing the size distribution of the DNA molecules Within each sample and selecting a set of samples based on the size distri -
`bution of the DNA molecules Within said samples; - performing a massively parallel sequencing of DNA of each size-selected
`sample; — mapping the obtained sequences to the human genome for each sample; — calculating a set of reference parameters, wherein
`each reference parameter is indicative of the number of unique exact sequences mapped to a chromosome or chromosomal region of
`interest for each sample; — obtaining a set ofreferenee samples and/or a set of reference parameters.
`
`
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`

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`WO 2014/068075
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`PCT/EP2013/072848
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`Non-invasive method for detecting a fetal chromosomal aneuploidy
`
`The present invention relates to non-invasive prenatal diagnosis of fetal aneuploidy using
`
`cell-free DNA, particularly size-selected cell-free DNA. More particularly,
`
`the invention
`
`relates to methods of diagnosis of fetal aneuploidy characterized by the use of a set of
`
`external
`
`reference samples providing highly improved sensitivity and specificity. The
`
`invention also relates to methods for obtaining the reference samples and kits comprising
`
`the reference samples and / or a set of reference parameters for use in diagnosis of fetal
`
`aneuploidy.
`
`The detection of fetal chromosomal aneuploidies is an important procedure in prenatal
`
`diagnosis. Several major diseases are caused by chromosomal aneuploidies, such as
`
`Down syndrome (also referred to as trisomy 21), trisomy 18, trisomy 13, and it is of utmost
`
`importance to predict as soon as possible whether a fetus will be affected by one of these
`
`anomalies. Moreover,
`
`the risk that a fetus will be afflicted by an aneuploidy generally
`
`increases with the mother’s age. Therefore, the increase in the average age of pregnant
`
`women in most developed countries further raises the need for powerful and safe
`
`diagnostic methods for detecting fetal chromosomal aneuploidies.
`
`The detection of fetal chromosomal aneuploidies is commonly performed through invasive
`
`procedures such as chorionic villus sampling, amniocentesis or cord blood sampling.
`
`These methods have in common that they rely on the collection of a fetal biological
`
`material (amniotic fluid, chorionic villi, cord blood) in order to obtain fetal cells, necessary
`
`for a karyotype analysis. These methods have been routinely practised for a long time.
`
`However, due to their invasiveness, they are not free of risk for the fetus and for the
`
`mother. The most frequent risk is the chance of miscarriage, close to 1% in the case of
`
`amniocentesis. Other risks are associated with these invasive procedures, such as risks of
`
`infection, transmission of a disease from the mother to the fetus (for example AIDS or
`
`hepatitis B), amniotic fluid leakage, or premature birth.
`
`Non-invasive methods based on ultrasound scanning or on the detection of maternal
`
`serum biochemical markers have also been developed, but these methods are mainly
`
`restricted to the detection of epiphenomena, and have a limited clinical usefulness for
`
`detecting the core pathologies of chromosomal abnormalities.
`
`

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`WO 2014/068075
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`PCT/EP2013/072848
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`The discovery of cell—free fetal nucleic acids in maternal plasma in 1997 opened up new
`
`possibilities. The first strategies using these nucleic acids for assessing the fetal
`
`chromosomal dosage were based on the analysis of the allelic ratio of SNPs in target
`
`nucleic acids (placental mRNA and DNA molecules bearing a placental-specific DNA
`
`methylation signature) based on the assessment of the fetal chromosomal dosage by
`
`allelic ratio analysis of SNPs. Another strategy was developed more recently using digital
`
`PCR (Lo et al., 2007). The technique consists in measuring the total amount of a specific
`
`locus on a potentially aneuploid chromosome (for example chromosome 21) in maternal
`
`plasma and comparing this amount to that on a reference chromosome.
`
`In 2008, Chiu et al successfully implemented massively parallel sequencing in a method
`
`for diagnosing fetal
`
`trisomy 21 in maternal plasma (Chiu et al., 2008). Their method
`
`consists in performing a massively parallel sequencing on DNA extracted from the plasma
`
`samples. The sequences obtained from the MPGS step are then aligned to a reference
`
`sequence of the human genome, and the number of sequences which have been uniquely
`
`mapped to a location on the human genome, without mismatch,
`
`is counted for each
`
`chromosome, and compared to the total number of sequences obtained during the MPGS.
`
`This ratio provides an indication of
`
`the “chromosomal
`
`representation” of
`
`the DNA
`
`molecules found in a maternal plasma sample. The overrepresentation of chromosome 21
`
`in a given sample, by comparison to a set of reference samples already known as eu ploid,
`
`is indicative of a fetal trisomy 21.
`
`Approximately at the same time, Fan et al successfully developed another method for the
`
`diagnosis of fetal trisomy 21, using shotgun sequencing of cell-free plasma (Fan et al.,
`
`2008). After massively sequencing the cell-free DNA extracted from maternal plasma
`
`samples, Fan et al. mapped each sequence to the human genome. Each chromosome of
`
`the human genome was then divided into 50 kb bins, and, for each bin the number of
`
`sequence tags uniquely mapped to the human genome with at most one mismatch was
`
`counted. Fan et al. then calculated the median value of this count of sequence tag over
`
`each chromosome. Finally, Fan et al. compared the chromosome 21 sequence tag density
`
`of plasma issued from mothers carrying a fetus afflicted by trisomy 21 to that of plasma
`
`issued from mothers carrying euploid fetuses, and they noticed that
`
`the trisomy 21
`
`sequence tag density was higher than that of euploid samples, with a 99% confidence
`level.
`
`

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`WO 2014/068075
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`PCT/EP2013/072848
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`These techniques both rely on the detection of the overrepresentation of a given
`
`chromosome in comparison to euploid reference samples. They have provided a useful
`
`“proof-of—concept” and have paved the way for an efficient use of next-generation
`
`sequencing technology in the diagnosis of fetal aneuploidy. However, the implementation
`
`of the method in a routine clinical context requires a higher level of sensitivity and
`
`specificity than that currently described in the prior art.
`
`The sensitivity of non-invasive prenatal diagnosis to detect fetal aneuploidy with whole
`
`genome next generation sequencing (WG-NGS) depends on the fetal DNA fraction in the
`
`maternal plasma, and on the sequencing depth. While the fetal DNA fraction depends on a
`
`series of
`
`largely inherent biological variables,
`
`the technical variables
`
`subject
`
`to
`
`experimental modification include i), the efficiency of the DNA extraction procedure, ii), the
`
`accuracy and throughput of NGS, namely the fraction of sequence tags with unique exact
`
`matches that can be aligned to the sequenced genome (termed “unique exact sequences
`
`without mismatches" or “UES”) and the total number of molecules sequenced iii),
`
`the
`
`nature of the bioinformatic algorithms, and iv), the control group of samples from pregnant
`
`women with normal fetal caryotypes that provides the reference set. The latter is of utmost
`
`importance, since individual molecules counting for each single chromosome is normalized
`
`with the median sequence tag density of all autosomes (Fan et al 2008).
`
`The present invention implements a DNA extraction method not previously used for non-
`
`invasive prenatal diagnosis and having a fivefold greater yield than standard methods,
`
`together with a rigorously quality-controlled NGS work-flow with overall 25-30% more
`
`UESs than the published references, and average total count of UESs of more than
`
`15-106, which is three times higher than the current standard. The final readout of the test
`
`fits the requirements of a robust clinical test, is. a 100% sensitivity and 100% specificity for
`
`the major fetal aneuploidies. This procedure for instance discriminates trisomy 21 or Down
`
`syndrome from normal male and female caryotypes with 51.1-10'5 prior probability of
`
`generating false results by chance. Since the benchmark is 52.7-10'3,
`
`it represents an
`
`improvement of
`
`two orders of magnitude. This invention provides a combination of
`
`methods that allow the constitution of a high quality reference set of sequences, which is
`
`the key step towards defining the performance of the NGS procedure.
`
`

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`WO 2014/068075
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`PCT/EP2013/072848
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`A first aspect of the present invention thus relates to a method for obtaining a set of
`
`reference samples and/or a set of reference parameters for
`
`the diagnosis of fetal
`
`aneuploidy from a maternal biological sample, preferably a blood sample, comprising:
`
`a step of extracting cell-free DNA from a set of biological samples, preferably blood
`
`samples, obtained from euploid pregnant women carrying a euploid fetus;
`
`a step of performing a massively parallel sequencing of DNA of each sample;
`
`a step of mapping the obtained sequences to the human genome for each sample;
`
`optionally calculating a set of
`
`reference parameters, wherein each reference
`
`parameter is
`
`indicative of the number of unique exact sequences mapped to a
`
`chromosome or chromosomal region of interest for each sample;
`
`obtaining a set of reference samples and/or a set of reference parameters;
`
`wherein the method comprises at least one of the following additional steps/features:
`
`the extraction of cell-free DNA from each biological sample comprises:
`
`0 mixing said biological sample with a composition comprising chloroform and
`
`phenoh
`
`o
`
`0
`
`0
`
`extracting the aqueous phase from said mixture;
`
`precipitating DNA from said aqueous phase;
`
`optionally collecting precipitated DNA.
`
`After the extraction step, analyzing the size distribution of the DNA molecules within
`
`each sample and selecting a set of samples based on the size distribution of the DNA
`
`molecules within said samples;
`
`After the extraction step or after the selection step based on the size distribution of the
`
`DNA molecules, pre-sequencing DNA of each sample, mapping the obtained
`
`sequences to the human genome, and selecting a set of samples based on the
`
`amount of unique exact sequences mapped to the human genome;
`
`After the step of mapping the sequences obtained from massively parallel sequencing,
`
`selecting a set of samples based on the number of unique exact sequences mapped to
`
`the human genome.
`
`The method can comprise any one of these additional steps or features, any combination
`
`of two or three of these additional steps or features or the four additional steps and
`featu res.
`
`Preferably, the method of the invention includes a step of size selection of the cell-free
`
`DNA, particularly immediately after the extraction step and prior to massive parallel
`
`

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`WO 2014/068075
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`PCT/EP2013/072848
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`sequencing. According to this embodiment, the invention relates to a method for obtaining
`
`a set of reference samples and/or a set of reference parameters for the diagnosis of fetal
`
`aneuploidy from a maternal biological sample, containing cell-free DNA, said method
`
`comprising:
`
`-
`
`-
`
`-
`
`extracting cell-free DNA from a set of biological samples obtained from euploid
`
`pregnant women carrying a euploid fetus;
`
`after the extraction step, analyzing the size distribution of the DNA molecules within
`
`each sample and selecting a set of samples based on the size distribution of the DNA
`
`molecules within said samples;
`
`performing a massively parallel sequencing of DNA of each size-selected sample;
`
`- mapping the obtained sequences to the human genome for each sample;
`
`—
`
`-
`
`calculating a set of reference parameters, wherein each reference parameter is
`
`indicative of the number of unique exact sequences mapped to a chromosome or
`
`chromosomal region of interest for each sample;
`
`obtaining a set of reference samples and/or a set of reference parameters.
`
`A preferred example of such a method for obtaining a set of reference samples, including a
`
`size-selection step, comprises :
`
`a) extracting cell-free DNA from a set of biological samples obtained from euploid pregnant
`
`women carrying a euploid fetus, and optionally also obtained from euploid pregnant
`
`women carrying an aneuploid fetus;
`
`b) subjecting the samples of extracted cell—free DNA to a step of size selection, particularly
`
`to remove cell-free DNA molecules having a size greater than 200 bp;
`
`0) processing the size-selected extracted DNA samples obtained in step (b) for the
`
`preparation of a sequencing library, for example by end repair of the DNA molecules and
`
`ligation of sequencing adaptors, optionally followed by amplification of the adaptor-ligated
`
`fragments;
`
`d) performing a massively parallel sequencing of DNA of each size—selected sample
`
`obtained in (c);
`
`e) mapping the sequences obtained in step (d) to the human genome for each sample;
`
`f) calculating a set of reference parameters, wherein each reference parameter is
`
`indicative of the number of unique exact sequences mapped to a chromosome or
`
`chromosomal region of interest for each sample;
`
`9) obtaining a set of reference samples and/or a set of reference parameters.
`
`

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`WO 2014/068075
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`PCT/EP2013/072848
`
`It
`
`is particularly preferred that,
`
`in obtaining the reference set of samples,
`
`the set of
`
`biological samples from which cell-free DNA is extracted further includes samples obtained
`
`from euploid pregnant women carrying an aneuploid fetus,
`
`In this way, the reference set
`
`provides reference values for both euploid and aneuploid samples.
`
`In an alternative embodiment, the method for obtaining a set of reference samples for the
`
`diagnosis of fetal aneuploidy from a maternal biological sample containing cell-free DNA,
`
`comprises steps of pre-sequencing and mapping on a size-selected sub-set of samples
`
`prior to massive parallel sequencing. According to this alternative embodiment the method
`
`comprises:
`
`(i)
`
`extracting cell-free DNA from a set of biological samples, preferably blood
`
`samples, obtained from a set of euploid pregnant women carrying a euploid
`
`fetus;
`
`(ii)
`
`(iii)
`
`(iv)
`
`(v)
`
`(vi)
`
`analyzing the size distribution of the DNA molecules within each sample;
`
`selecting a first set of samples based on the size distribution of the DNA
`
`molecules within said samples;
`
`pre-sequencing DNA of each sample from said first set of samples;
`
`mapping the sequences obtained in step (iv) to the human genome;
`
`selecting a second set of samples based on the amount of unique exact
`
`sequences mapped to the human genome in step (V);
`
`(vii)
`
`massively parallel sequencing DNA of each sample from said second set of
`
`samples;
`
`(viii)
`
`mapping the sequences obtained in step (vii) to the human genome;
`
`(ix)
`
`selecting a set of reference samples based on the number of unique exact
`
`sequences mapped to the human genome in step (viii).
`
`In a specific embodiment, step (iii) comprises selecting samples in which at least 90 wt%,
`
`preferably more than 95wt% of the DNA molecules have a size from 156 bp to 176 bp.
`
`In another embodiment, step(iii) comprises selecting samples with at least 0.88 ng/ul DNA
`
`molecules with a size from 156 bp to 176 bp.
`
`In another embodiment, step (iv) comprises sequencing from 1000 to 100000 sequences
`
`within each sample.
`
`

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`WO 2014/068075
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`PCT/EP2013/072848
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`In another embodiment, step (vi) comprises selecting samples having at least 70 % of
`
`unique exact sequences with respect to the total number of sequences obtained in step
`
`(iv).
`
`In another embodiment, step (vii) comprises sequencing at least 25 million sequences for
`
`each sample. In another embodiment, step (vii) comprises obtaining at least 25 million filter
`
`passing reads for each sample.
`
`In another embodiment, step (ix) comprises selecting samples having more than 15
`
`millions unique exact sequence reads.
`
`The present invention also relates to a method for diagnosing fetal aneuploidy from a
`
`maternal biological test sample, preferably a blood sample, comprising:
`
`(a) extracting cell-free DNA from a maternal biological test sample obtained from a
`
`pregnant woman;
`
`(b) massively parallel sequencing cell-free DNA extracted from said test sample;
`
`(c) mapping the sequences obtained in step (b) to the human genome;
`
`(d) calculating a test parameter indicative of the number of unique exact sequences
`
`mapped to a chromosome or chromosomal region of interest;
`
`(e) calculating a set of reference parameters, wherein each reference parameter is
`
`indicative of the number of unique exact sequences mapped to a chromosome or
`
`chromosomal region of interest for a sample of a set of reference samples, such
`
`as a set of euploid reference samples, for example as obtained according to the
`
`present invention;
`
`(f) Comparing said test parameter calculated in step (d) with said set of reference
`
`parameters calculated at step (e);
`
`(9) based on the comparison, diagnosing a fetal aneuploidy.
`
`A preferred method of diagnosis of fetal aneuploidy comprises the above method in which,
`
`after the extraction step, a step of size selection based on the size of the DNA molecules
`
`within said sample is carried out. The step of size selection substantially eliminates DNA
`
`molecules having a size greater than 200 bp from the test sample. This step is preferably
`
`conducted prior to the preparation of a sequencing library. This method of diagnosis is
`
`particularly preferred in conjunction with the use of reference samples which have also
`
`undergone a step of cell-free DNA size selection as described above. Indeed, according to
`
`

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`WO 2014/068075
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`PCT/EP2013/072848
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`the invention,
`
`it is preferred that the test sample be subject to the same methodology as
`
`the reference samples.
`
`According to this preferred embodiment, the method for diagnosing fetal aneuploidy from a
`
`maternal biological test sample, preferably a blood sample, comprises:
`
`(a)
`
`extracting cell—free DNA from a maternal biological test sample such as blood
`
`obtained from a pregnant woman;
`
`(b)
`
`performing a step of size selection on the extracted cell-free DNA, such that DNA
`
`molecules having a size greater than 200 bp are substantially eliminated from the
`
`sample ;
`
`processing the size-selected extracted cell-free DNA for the preparation of a
`
`sequencing library, for example by end repair of the DNA molecules and ligation of
`
`sequencing adaptors, optionally followed by amplification of the adaptor-ligated
`
`fragments;
`
`(d)
`
`massively parallel sequencing the cell-free DNA obtained in step (c);
`
`(e)
`
`mapping the sequences obtained in step (d) to the human genome;
`
`(f)
`
`calculating a test parameter indicative of the number of unique exact sequences
`
`mapped to a chromosome or chromosomal region of interest;
`
`(9)
`
`calculating a set of reference parameters, wherein each reference parameter is
`
`indicative of the number of unique exact sequences mapped to a chromosome or
`
`chromosomal region of interest for a sample of a set of reference samples, such
`
`as a set of euploid reference samples, obtained according to the size—selection
`
`method of the present invention;
`
`(h)
`
`Comparing said test parameter calculated in step (f) with said set of reference
`
`parameters calculated at step (9);
`
`(i)
`
`based on the comparison, diagnosing a fetal aneuploidy.
`
`Preferably,
`
`the extraction of cell—free DNA from the maternal biological
`
`test sample
`
`comprises:
`
`mixing said biological sample with a composition comprising chloroform and
`
`phenol;
`
`extracting the aqueous phase from said mixture;
`
`precipitating DNA from said aqueous phase;
`
`optionally collecting precipitated DNA.
`
`

`

`WO 2014/068075
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`PCT/EP2013/072848
`
`In a specific embodiment, said test parameter is the unique sequence tag density of the
`
`chromosome or chromosomal region of interest normalized to the median unique exact
`
`sequence tag density of all autosomes.
`
`In another embodiment, said test parameter is the percentage of unique exact sequences
`
`mapped to said chromosome or chromosomal region, with respect to the total number of
`
`unique exact sequences mapped to all chromosomes, or to the total number of unique
`
`exact sequences mapped to all autosomes.
`
`In another embodiment, the comparison in step (f) is made through calculation of the z-
`
`score of said test parameter with respect to the set of reference parameters.
`
`In another embodiment, the test parameter is the absolute exact sequence count for the
`
`chromosome or chromosomal region of interest or the average exact sequence count for
`
`the chromosome or chromosomal region of interest.
`
`In a further embodiment the comparison in step (f) is made through calculation of the
`
`probability that the unique exact sequence count for the chromosome or chromosomal
`
`region of
`
`interest, or
`
`the average exact sequence count
`
`for
`
`the chromosome or
`
`chromosomal region of interest, belongs to the normal distribution of the unique exact
`
`sequence counts for the chromosome of interest of the reference set.
`
`In another embodiment, the chromosome of interest is chromosome 21, chromosome 18,
`
`chromosome 16, chromosome 11 or chromosome 13.
`
`In another embodiment, the chromosome of interest is chromosome 21, and the z-score of
`
`a trisomy 21 sample is at least 4.4 while the absolute value of the z—score of a sample
`
`euploid for chromosome 21 is less than 4.4.
`
`The present invention also relates to a method for extracting cell-free DNA from a maternal
`
`biological sample containing fetal and maternal cell-free DNA, comprising:
`
`-
`
`-
`
`-
`
`mixing said biological sample with a composition comprising chloroform and
`
`phenoh
`
`extracting the aqueous phase from said mixture;
`
`precipitating DNA from said aqueous phase;
`
`

`

`WO 2014/068075
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`PCT/EP2013/072848
`
`—
`
`optionally collecting precipitated DNA.
`
`The present invention also relates to the use of chloroform and phenol, preferably of a
`
`composition comprising chloroform and phenol for extracting cell-free DNA from a maternal
`
`biological sample containing fetal and maternal cell-free DNA.
`
`In a specific aspect, said use is in a method for obtaining a set of reference samples for the
`
`diagnosis of fetal aneuploidy from a maternal biological sample.
`
`In another aspect, said use is in a method for diagnosing fetal aneu ploidy from a maternal
`
`biological test sample
`
`The present invention also relates to a set of reference samples obtainable according to
`
`the method of the present invention.
`
`The present invention also relates to a computer program product for implementing one or
`
`more steps of the method for obtaining a set of reference samples for the diagnosis of fetal
`
`aneuploidy from a maternal biological sample.
`
`The present invention also relates to a computer program product for implementing one or
`
`more steps of the method for diagnosing fetal aneuploidy from a maternal biological test
`
`sample, for example one or more of step (d) to (g).
`
`The present invention also relates to a kit comprising one or more of:
`
`-
`
`-
`
`—
`
`-
`
`-
`
`one or more compositions and/or a kit for extracting cell-free DNA, for example
`
`including a composition comprising phenol and chloroform;
`
`a set of reference samples obtainable according to the method of the present
`
`invention;
`
`a set of reference parameters obtainable according to the method according to the
`
`present invention, optionally included in a physical support, such as a computer
`
`readable media;
`
`a computer program product for implementing one or more steps of the method for
`
`obtaining a set of reference samples for the diagnosis of fetal aneuploidy from a
`
`maternal biological sample;
`
`a computer program product for implementing one or more steps of the method for
`
`diagnosing fetal aneuploidy from a maternal biological test sample.
`
`10
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`WO 2014/068075
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`PCT/EP2013/072848
`
`According to a preferred embodiment,
`
`the kit
`
`for
`
`the diagnosis of fetal aneuploidy
`
`comprises :
`
`-
`
`a set of reference samples obtainable according to the method of the invention, for
`
`example a set of samples having undergone size selection to enrich the sample for
`
`cell—free DNA having a size of s 200bp, and eliminating DNA molecules greater
`
`than 200 bp, and comprising not only samples from euploid pregnant women
`
`carrying a euploid fetus but also samples from euploid pregnant women carrying
`
`an aneuploid fetus
`
`-
`
`and / or a set of reference parameters wherein each reference parameter is
`
`indicative of the number of unique exact sequences mapped to a chromosome or
`
`chromosomal
`
`region of
`
`interest for a sample of a reference set obtainable
`
`according to the method of the invention, optionally included in a physical support,
`
`Such a kit may further comprise at least one of :
`
`-
`
`-
`
`-
`
`one or more compositions and/or a kit for extracting cell-free DNA,
`
`including a
`
`composition comprising phenol and chloroform;
`
`a computer program product for implementing one or more steps of the method for
`
`obtaining a set of reference samples for the diagnosis of fetal aneuploidy from a
`
`maternal biological sample;
`
`a computer program product for implementing one or more steps of the method for
`
`diagnosing fetal aneuploidy from a maternal biological test sample.
`
`Brief Description of the drawings
`
`Figure 1:
`
`size distribution of 3 maternal plasma samples as obtained by capillary
`
`electrophoresis. The DNA molecules in these samples are ligated to a 132 bp sequencing
`
`adaptor/barcode.
`
`Figure 2: total number of filter passing sequence reads obtained by NGS sequencing for
`
`91 samples (eu ploid and aneuploid). The axis legend in ordinate reads “Cnt +1e6”, namely
`
`the sequence count in million.
`
`Figure 3: number of unique exact sequences for the same samples shown in Fig. 2. The
`
`axis legend in ordinate reads “Cnt +1 e6”, namely the sequence count in million.
`
`Figure 4: percentage of total unique sequence reads mapped to chromosome 21 with
`
`1/100,000 confidence interval (z-score=4.4) with respect to known healthy individuals
`
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`PCT/EP2013/072848
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`(reference samples selected according to the method of the present
`
`invention). The
`
`horizontal middle dotted line corresponds to the mean percentage of the reference sample.
`
`The horizontal full lines above and below the dotted line correspond to the discrimination
`
`threshold (mean i 4.4* SD). The trisomy 21 samples are positively discriminated.
`
`Figure 5: percentage of total unique sequence reads mapped to chromosome 18 with
`
`1/100,000 confidence interval
`
`(z—score=4.4) with respect to known healthy individuals
`
`(reference samples selected according to the method of the present
`
`invention). The
`
`horizontal middle dotted line corresponds to the mean percentage of the reference sample.
`
`The horizontal full lines above and below the dotted line correspond to the discrimination
`
`threshold (mean i 4.4* SD). The trisomy 18 samples are posititively discriminated.
`
`Figure 6: Scores of chromosome 1 using a second scoring algorithm. The discrimination
`
`thresholds correspond to a 1/100,000,000,000 confidence interval with respect to known
`
`healthy individuals (reference samples selected according to the method of the present
`
`invention).
`
`Figure 7: Scores of chromosome 19 score using a second scoring algorithm. The
`
`discrimination thresholds correspond to a 1/100,000,000,000 confidence interval with
`
`respect to known healthy individuals (reference samples selected according to the method
`
`of the present invention).
`
`Figure 8: Scores of chromosome 13 score using a second scoring algorithm. The
`
`discrimination thresholds correspond to a 1/100,000,000,000 confidence interval with
`
`respect to known healthy individuals (reference samples selected according to the method
`
`of the present invention). The trisomy 13 sample is positively discriminated.
`
`Figure 9: Scores of chromosome 18 using a second scoring algorithm. The discrimination
`
`thresholds correspond to a 1/100,000,000,000 confidence interval with respect to known
`
`healthy individuals (reference samples selected according to the method of the present
`
`invention). The trisomy 18 samples are positively discriminated.
`
`Figure 10: Scores of chromosome 21
`
`using a second scoring algorithm. The
`
`discrimination thresholds correspond to a 1/100,000,000,000 confidence interval with
`
`respect to known healthy individuals (reference samples selected according to the method
`
`of the present invention). The trisomy 21 samples are positively discriminated.
`
`Figure 11: Scores of chromosome 22 using a second scoring algorithm. The
`
`discrimination thresholds correspond to a 1/100,000,000,000 confidence interval with
`
`respect to known healthy individuals (reference samples selected according to the method
`
`of the present invention). The trisomy 22 sample is positively discriminated.
`
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`Figure 12: Scores of chromosome 4 using a second scoring algorithm. The discrimination
`
`thresholds correspond to a 1/100,000,000,000 confidence interval with respect to known
`
`healthy individuals (reference samples selected according to the method of the present
`
`invention). The 4p microdeletion (Wolf-Hirschhorn syndrome) sample is negatively
`discriminated.
`
`Figure 13: Scores of chromosome 5 using a second scoring algorithm. The discrimination
`
`thresholds correspond to a 1/100,000,000,000 confidence interval with respect to known
`
`healthy individuals (reference samples selected according to the method of the present
`
`invention). The 5p microdeletion/duplication (cri du chat syndrome) sample is positively
`discriminated.
`
`Figure 14: Sequence tag densities over chromosome 4 of a 4p microdeletion syndrome
`
`sample. A negative deviation from the mean density of the reference samples is apparent
`
`at the location of the 4p deletion.
`
`Figure 15: Sequence tag densities over chromosome 5 of a 5p microdeletion/duplication
`
`syndrome sample. Positive and negative deviations from the mean density of the reference
`
`samples are apparent at the location of the 5p microdeletion and duplication, respectively.
`
`The data shown on Figures 2 to 13 were all obtained with the same set of 91 samples, and
`
`are