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WO2023116717A1 - Procédé de surveillance d'une fraction d'adn donneur - Google Patents

Procédé de surveillance d'une fraction d'adn donneur Download PDF

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WO2023116717A1
WO2023116717A1 PCT/CN2022/140435 CN2022140435W WO2023116717A1 WO 2023116717 A1 WO2023116717 A1 WO 2023116717A1 CN 2022140435 W CN2022140435 W CN 2022140435W WO 2023116717 A1 WO2023116717 A1 WO 2023116717A1
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transplant
recipient
sample
cfdna
donor
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Jianxing HE
Liping Liu
Liyan Huang
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First Affiliated Hospital of Guangzhou Medical University
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First Affiliated Hospital of Guangzhou Medical University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • Organ transplantation is now a preferred practice for patient with end-stage organ failure.
  • the clinical outcomes remain poor with median survival rate.
  • the immunological reaction of rejection is a major cause of functional failure in transplant patients.
  • Accurate monitoring of graft is essential for long-term survival of the transplant recipient.
  • the current "gold standard" for detecting or confirming graft rejection following organ transplantation requires biopsy samples (for example, transbronchial biopsy for lung transplant and endomyocardial biopsy for heart transplant) in order to detect immune cell (e.g., T-cells, macrophages, etc. ) infiltration into the graft and other pathological changes.
  • immune cell e.g., T-cells, macrophages, etc.
  • donor DNA fraction fraction of donor-derived cell-free DNA (cfDNA) in the recipient’s plasma
  • donor DNA fraction fraction of donor-derived cell-free DNA
  • previous studies mainly focused on a global estimation of donor DNA fraction conducted months post-transplant, and there is a critical need for non-invasive detection methods that can be used in early detection of allograft rejection and prognosis.
  • the present invention relates to a method for detecting donor cfDNA fraction in a sample of a recipient.
  • the present invention further relates to a method for detecting over-represented SNP (s) of donor cfDNA in an identified region.
  • the methods of the present invention may be used to establish the role of early donor DNA dynamics on post-transplantation manipulation, or provide clinical data for determining the likelihood of transplant rejection, in particular, acute rejection, or monitor immunosuppressive therapy in a subject.
  • the present invention relates to a method for detecting donor cfDNA fraction in a sample of a transplant recipient, comprising:
  • extracting DNA from the sample of the transplant recipient comprises extracting genomic DNA from the sample of the recipient pre-transplant.
  • cfDNA is extracted from the sample of the recipient post-transplant.
  • cfDNA is extracted from the sample of the recipient post-transplant, preferably, during the first four weeks post-transplant, preferably three weeks post-transplant, more two weeks post-transplant.
  • cfDNA is extracted from the sample of the recipient on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 post-transplant.
  • the method comprises a step of preparing a library of the extracted DNA from the sample of the transplant recipient.
  • the method comprises a step of preparing a library of the genomic DNA extracted from the sample of the recipient pre-transplant.
  • the genomic DNA is fragmented into 100bp-300bp size in length, more preferably, ⁇ 250bp size in length.
  • the method comprises a step of preparing a library of cfDNA extracted from the sample of the recipient post-transplant.
  • cfDNA is 60bp ⁇ 200bp size in length.
  • the method further comprises a step of sequencing DNA of the prepared library.
  • sequencing adaptors are ligated to the fragmented DNA or cfDNA.
  • the step of preparing the library universal amplification is performed for the extracted DNA.
  • the amplification product is sequenced by high-throughput sequencing.
  • end repair is performed for cfDNA.
  • dA-tailing process is performed for the fragmented DNA, or cfDNA. Specifically, a non-template dAMP is added onto 3’ end of the fragmented DNA, or cfDNA.
  • the method comprises a step of selecting the genome wide SNPs that have a homozygous genotype in the sample of the recipient pre-transplant, and a different genotype in the sample of the recipient post-transplant. SNP is called if the read genotype differs between the sample of the recipient pre-transplant and the sample of the recipient post-transplant.
  • the method comprises a step of calculating the minor allele frequency (MAF) for each of the selected SNPs.
  • MAF minor allele frequency
  • the donor cfDNA fraction is an average of all observed values of donor fraction (herein denoted as ⁇ ) deducted from each selected SNP i .
  • the donor cfDNA fraction is calculated utilizing the following formula 1:
  • ⁇ is the average of the list of MAFs, which is a mixture of two normal distributions, one centered at 0.5 ⁇ and the other centered at ⁇ , and 0.5 ⁇ ⁇ ⁇ , which transforms into:
  • the donor genotype on a given SNP i is also homozygous (i.e., wildtype/wildtype in recipient and mutant/mutant in donor or the other way around) , ⁇ i is equal to MAF; if the donor genotype is heterozygous, ⁇ i will equal two times MAF (i.e., half of the donor fraction equals MAF) .
  • k-means clustering does not guarantee a global optimum (can be trapped into local optima)
  • All values in the sorted list before the best separating value point are considered as samples of the 0.5 ⁇ distribution, these values are then multiplied by 2.
  • the mean of the resulted new list of values is the predicted ⁇ , i.e., the predicted global donor DNA fraction.
  • the donor cfDNA fraction of greater than 1% indicates that the transplant is undergoing acute rejection, and the donor cfDNA fraction of less than 1%indicates that the transplant is undergoing borderline rejection, undergoing other injury, or stable.
  • the method further comprises a step of determining the occurrence or likely occurrence of active rejection of transplantation using the detected donor cfDNA fraction in the sample of the transplant recipient.
  • the sample is a blood sample, a urine sample, or a body fluid sample.
  • the transplant recipient is a mammal, preferably, a human.
  • the transplant recipient received a transplant selected from organ transplant, tissue transplant, and cell transplant.
  • the transplant recipient received a transplant selected from, but not limited to, lung transplant, kidney transplant, heart transplant, liver transplant, pancreas transplant, intestinal transplant, stomach transplant, testis transplant, penis transplant, thymus transplant, uterus transplant, bone transplant, bone marrow transplant, tendon transplant, cornea transplant, uterus transplant, ovary transplant, nerve transplant, pancreas islet cell transplant, blood vessel transplant, heart valve transplant, skin transplant, hand transplant, and/or leg transplant.
  • a transplant selected from, but not limited to, lung transplant, kidney transplant, heart transplant, liver transplant, pancreas transplant, intestinal transplant, stomach transplant, testis transplant, penis transplant, thymus transplant, uterus transplant, bone transplant, bone marrow transplant, tendon transplant, cornea transplant, uterus transplant, ovary transplant, nerve transplant, pancreas islet cell transplant, blood vessel transplant, heart valve transplant, skin transplant, hand transplant, and/or leg transplant.
  • the present invention relates to a method for detecting over-represented donor cfDNA in a sample of a transplant recipient, comprising:
  • genomic DNA is extracted from the sample of the recipient pre-transplant.
  • cfDNA is extracted from the sample of the recipient post-transplant.
  • cfDNA is extracted from the sample of the recipient during the first two weeks post-transplant.
  • cfDNA is extracted from the sample of the recipient on day 1, 4, 7, 10, or 13 post-transplant.
  • the method comprises a step of preparing a library of the extracted DNA from the sample of the transplant recipient.
  • the method comprises a step of preparing a library of the genomic DNA extracted from the sample of the recipient pre-transplant.
  • the genomic DNA is fragmented into 200bp-300bp size in length, more preferably, ⁇ 250bp size in length.
  • the method comprises a step of preparing a library of cfDNA extracted from the sample of the recipient post-transplant.
  • cfDNA is 60bp ⁇ 200bp size in length. More preferably, cfDNA is ⁇ 170bp size in length.
  • the method further comprises a step of sequencing DNA of the prepared library.
  • sequencing adaptors are ligated to the fragmented DNA or cfDNA.
  • the step of preparing the library universal amplification is performed for the extracted DNA.
  • the amplification product is sequenced by high-throughput sequencing.
  • end repair is performed for cfDNA.
  • dA-tailing process is performed for the fragmented DNA, or cfDNA. Specifically, a non-template dAMP is added onto 3’ end of the fragmented DNA, or cfDNA.
  • the step of calculating donor cfDNA fraction based on SNP genotyping comprises splitting chromosome into windows covering 100-1000kb, preferably, 400-600kb, more preferably, 500kb in size, and calculating donor cfDNA fraction utilizing maximum-likelihood-estimation (MLE) .
  • MLE maximum-likelihood-estimation
  • 22 autosomes are splitted into windows (regions) covering 100-1000kb seperately, preferably, 400-600kb, more preferably, 500kb in size.
  • the step of calculating donor cfDNA fraction comprises calculating p-value for each window based on assuming normal distribution of the donor cfDNA fraction values, wherein for each SNP (denoted as SNP i ) , p i -value can be calculated as follows:
  • the total probability p is the products of each p i :
  • each SNP has only three possible values of wildtype frequency: ⁇ 0, 0.5, 1 ⁇ on both the recipient and the donor;
  • the maximum log p for the given ⁇ can be calculated. Different choices of ⁇ values yield different maximum log p (i.e., log-likelihood) . By iterating through evenly spaced numbers within a specified interval of possible ⁇ values, the method can find out the closest approximation of the best ⁇ resulting in a maximum log-likelihood.
  • the step of calculating donor cfDNA fraction comprises correcting the median p-values for multiple tests.
  • the step of selecting over-represented donor cfDNA comprises selecting over-represented donor cfDNA having an FDR ⁇ 0.1 for each transplant recipient.
  • the method further comprises a step of selecting an over-represented region.
  • An over-represented region is defined as having an FDR ⁇ 0.1 for each transplant recipient.
  • the method further comprises a step of selecting over-represented SNP having an FDR ⁇ 0.1 for each transplant recipient.
  • the donor fraction ⁇ i for each selected SNP i is calculated according to formula 1 in the first place.
  • the method further comprises a step of selecting a region enrichment of significantly over-represented SNPs satisfying: 1) contain ⁇ 5 significant SNPs, and (2) each significant SNP is less than 0.5M apart from its nearest significant SNPs.
  • the over-represented regions are regions associated with graft-host immune responses and/or antimicrobial activities.
  • the over-represented regions are regions associated with HLA-A/B/C, MICA, DDR1, for example chr6: 30782303-31426881, and/or regions overlapped with the family of ⁇ defensin genes, e.g. DEFB103/104/105/106, for example, chr7: 75883092-77138957 and chr8: 7237702-7978545.
  • the sample is a blood sample, a urine sample, or a body fluid sample.
  • the transplant recipient is a mammal, preferably, a human.
  • the transplant recipient received a transplant selected from organ transplant, tissue transplant, and cell transplant.
  • the transplant recipient received a transplant selected from, but not limited to, lung transplant, kidney transplant, heart transplant, liver transplant, pancreas transplant, intestinal transplant, stomach transplant, testis transplant, penis transplant, thymus transplant, uterus transplant, bone transplant, bone marrow transplant, tendon transplant, cornea transplant, uterus transplant, ovary transplant, nerve transplant, pancreas islet cell transplant, blood vessel transplant, heart valve transplant, skin transplant, hand transplant, and/or leg transplant.
  • a transplant selected from, but not limited to, lung transplant, kidney transplant, heart transplant, liver transplant, pancreas transplant, intestinal transplant, stomach transplant, testis transplant, penis transplant, thymus transplant, uterus transplant, bone transplant, bone marrow transplant, tendon transplant, cornea transplant, uterus transplant, ovary transplant, nerve transplant, pancreas islet cell transplant, blood vessel transplant, heart valve transplant, skin transplant, hand transplant, and/or leg transplant.
  • the present invention relates to a method for determining the likelihood of transplant rejection in a transplant recipient, comprising the steps of the method in the first place, and/or the steps of the method in the second place.
  • the present invention relates to a method for predicting prognosis of transplant rejection in a transplant recipient, comprising the steps of the method in the first place, and/or the steps of the method in the second place.
  • the present invention relates to a method for diagnosing a transplant within a transplant recipient as undergoing acute rejection, comprising the steps of the method in the first place, and/or the steps of the method in the second place.
  • the present invention relates to an apparatus for detecting donor cfDNA fraction in a sample of a transplant recipient, for determining the likelihood of transplant rejection in a transplant recipient, for predicting prognosis of transplant rejection in a transplant recipient, or for diagnosing a transplant within a transplant recipient as undergoing acute rejection, comprising the following modules:
  • the apparatus further comprises a module for preparing a library of the extracted DNA from the sample of the transplant recipient.
  • the apparatus further comprises a module for preparing a library of the genomic DNA extracted from the sample of the recipient pre-transplant.
  • the apparatus further comprises a module for preparing a library of cfDNA extracted from the sample of the recipient post-transplant.
  • the apparatus further comprises a module for sequencing DNA of the prepared library.
  • the apparatus further comprises a module for universal amplification.
  • the apparatus further comprises a module for selecting the genome wide SNPs that have a homozygous genotype in the sample of the recipient pre-transplant, and a different genotype in the sample of the recipient post-transplant.
  • the present invention relates to an apparatus for detecting donor cfDNA fraction in a sample of a transplant recipient, for determining the likelihood of transplant rejection in a transplant recipient, for predicting prognosis of transplant rejection in a transplant recipient, or for diagnosing a transplant within a transplant recipient as undergoing acute rejection, comprising the following modules:
  • the apparatus further comprises a module for preparing a library of the extracted DNA from the sample of the transplant recipient.
  • the apparatus further comprises a module for preparing a library of the genomic DNA extracted from the sample of the recipient pre-transplant.
  • the apparatus further comprises a module for preparing a library of cfDNA extracted from the sample of the recipient post-transplant.
  • the apparatus further comprises a module for sequencing DNA of the prepared library.
  • the apparatus further comprises a module for universal amplification.
  • the present invention relates to use of an agent for detecting a region satisfying one of the following conditions: over-represented region having an FDR ⁇ 0.1 for each transplant recipient;
  • a region enrichment of significantly over-represented SNPs satisfying: 1) contain ⁇ 5 significant SNPs; and (2) each significant SNP is less than 0.5M apart from its nearest significant SNPs;
  • chr6 an over-represented region associated with HLA-A/B/C, MICA, DDR1, for example chr6: 30782303-31426881, and/or regions overlapped with the family of ⁇ defensin genes, e.g. DEFB103/104/105/106, for example, chr7: 75883092-77138957 and chr8: 7237702-7978545, for determining the likelihood of transplant rejection in a transplant recipient, for predicting prognosis of transplant rejection in a transplant recipient, or for diagnosing a transplant within a transplant recipient as undergoing acute rejection.
  • ⁇ defensin genes e.g. DEFB103/104/105/106, for example, chr7: 75883092-77138957 and chr8: 7237702-7978545, for determining the likelihood of transplant rejection in a transplant recipient, for predicting prognosis of transplant rejection in a transplant recipient, or for diagnosing a transplant within a transplant recipient as
  • the present invention relates to use of an agent for detecting donor cfDNA fraction in a sample of a transplant recipient for determining the likelihood of transplant rejection in a transplant recipient, for predicting prognosis of transplant rejection in a transplant recipient, or for diagnosing a transplant within a transplant recipient as undergoing acute rejection.
  • Figure 1A illustrates the study design and data analysis flowchart.
  • Figure 1B shows that the global donor DNA fraction for each transplant recipient at each time point.
  • Figure 2A shows shows log p-value vs chromosomal position of genome-wide SNPs for three recipients.
  • Figure 2B shows the defined significant regions according to the number of significant SNPs.
  • Figure 3A is a plot diagram showing hits with a p-value ⁇ 0.05.
  • Figure 3B shows graphs of relative coverage of several genes.
  • Figure 4 shows a circos plot of estimated regional donor DNA fraction on 500kb windows of patient 1.
  • Figure 5 shows a circos plot of estimated regional donor DNA fraction on 500kb windows of patient 2.
  • Figure 6 shows a circos plot of estimated regional donor DNA fraction on 500kb windows of patient 3.
  • Figure 7 shows that the over-representation is not likely to be caused by copy number variations (duplications) in the Dx samples.
  • Genome-wide SNPs (excluding sex chromosomes) that have a strictly homozygous genotype in the D0 (gDNA) sample and a different genotype in the Dx (cfDNA) samples are firstly selected. To reduce the effects of rare genetic variants on reproducibility, variants with a gnomAD_ALL population frequency lower than 0.3 are filtered [5] . For each of the selected SNPs, the minor allele frequency (MAF) is calculated [6, 7] .
  • the global donor DNA fraction is considered as an average of all observed values of donor fraction (herein denoted as ⁇ ) deducted from each selected SNP i . It is assumed that each ⁇ i follows normal distribution.
  • ⁇ i is equal to MAF; if the donor genotype is heterozygous, ⁇ i will equal two times MAF (i.e., half of the donor fraction equals MAF) . Therefore, the list of MAFs is a mixture of two normal distributions, one centred at 0.5 ⁇ and the other centred at ⁇ . Then the average of the list (denoted as x) is calculated and obtained 0.5 ⁇ ⁇ x ⁇ ⁇ , which transforms into
  • k-means clustering does not guarantee a global optimum (can be trapped into local optima)
  • All values in the sorted list before the best separating value point are considered as samples of the 0.5 ⁇ distribution, these values are then multiplied by 2.
  • the mean of the resulted new list of values is the predicted ⁇ , i.e., the predicted global donor DNA fraction.
  • the above genome-wide estimation method is not suitable for calculating donor DNA fraction in a small region (e.g. 500kb) , because there could be too few SNPs that satisfy the ‘strictly homozygous genotype in D0 sample and a different genotype in the Dx samples’ condition within a given small region.
  • a method that does not require D0 to be homozygous, which allows more SNP information to be considered is utilized. This is a simplified variant of a previously published maximum likelihood-based method [8] .
  • the total probability p is the products of each p i .
  • the regional method starts with all SNPs.
  • variants with a gnomAD_ALL population frequency lower than 0.01 are filtered. 22 autosomes are splitted into 500kb windows, and the donor DNA fraction for each region at each time point is estimated. Last window of each chromosome is less than 500kb; windows with no informative SNPs (i.e., differed D0 and Dx genotypes) are removed. For each transplant recipient, a circos plot of estimated regional donor DNA fraction is drawn for all time points ( Figures 4-6) .
  • donor fraction ⁇ i for each selected SNP i is estimated . Then a p-value for each ⁇ i assuming normal distribution is calculated, and the median p-values for multiple tests are corrected using Benjamini-Hochberg adjustment. Significantly over-represented SNP is defined as having an FDR ⁇ 0.1 for each transplant recipient.
  • a Manhattan plot of median log adjusted p-value of all selected SNPs over the genome (excluding sex chromosomes) is drawn for each recipient using the qqman R package [9] ( Figure 2A) .
  • Regions with enrichment of significantly over-represented SNPs is further defined as regions satisfying: (1) contain ⁇ 5 significant SNPs, and (2) each significant SNP is less than 0.5M apart from its nearest significant SNPs.
  • the identified enriched regions that overlapped in all recipients are highlighted in grey colour on the Manhattan plots.
  • Nucleosome foot-printing of genes of interest is performed as previously described [12, 13] .
  • a nucleosome footprint for a gene is the relative coverage (position depth divided by average depth) at ⁇ 1kb of the transcription start site (TSS) .
  • TSS transcription start site
  • the mean of the three D0 samples and mean of the total 15 Dx samples are used as input for plotting, to visualize changes of nucleosome occupancy pre-and post-transplant.
  • chr6 30782303-31426881
  • chr7 75883092-77138957
  • chr8 7237702-7978545
  • Significant regions are defined as having ⁇ 5 significant SNPs, each being less than 500kb apart from its closest significant SNPs ( Figure 2A and 2B) .
  • chr6 30782303-31426881 overlapped with the human leukocyte antigen (HLA) region, which includes a series of immune-related genes such as HLA-A/B/C, MICA, DDR1; chr8: 7237702-7978545 overlapped with the family of ⁇ defensin genes, e.g. DEFB103/104/105/106.
  • HLA human leukocyte antigen
  • nucleosome footprints of genes within the significant regions pre-and post-transplant are computed. Characteristics of open chromatin are found in some of the genes post-transplant, suggesting active transcriptions. Among these likely active genes, some are known to be expressed in lung epithelial during inflammation and transplantation, e.g. DEFB103, DDR1 and MICA; some known to be expressed in both the host and the graft, e.g. HLA-B/C. Interestingly, DEFB103 and DDR1, which are expected to be expressed only in the graft, showed different nucleosome footprints pre-and post-transplant, while genes known to express in both, e.g. HLA-B/C, preserved the shape of the footprints.
  • LTx lung transplantation
  • Genomic DNA is extracted from D0 samples and cfDNA is extracted from Dx samples.
  • purified gDNA from D0 samples is extracted using QIAamp DNA Blood Mini Kit (Qiagen, Germen) ;
  • cfDNA from Dx samples is extracted using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Germen) , according to the manufacturer’s instructions, separately.
  • gDNA preparation is performed with MGIEasy Universal DNA Library Prep Set and Customer self-developed kit (MGI, Shenzhen, China) .
  • MMI MGIEasy Universal DNA Library Prep Set and Customer self-developed kit
  • gDNA it is fragmented into ⁇ 250bp insert size length.
  • a non-templated dAMP is added onto the 3’ end of the fragments, which is known as dA-tailing.
  • cfDNA which is 60-200bp, for example, ⁇ 170bp, end repair and dA-tailing processes are performed.
  • Adapter ligation and Pre-PCR are performed, to obtain high-quality libraries before sequencing.
  • DNA quantification is performed with Qubit 3.0 (Thermo Fisher Scientific, USA) .
  • DNB preparation kits and Sequencing kits (MGI, Shenzhen, China) are used to pump DNBs (DNA nanoballs) and load the DNBs onto the Patterned Array chip. Pair-end, 150bp sequencing is performed using MGISEQ-2000 with an expected amount of data >150Gb per sample.
  • Raw sequencing reads are subjected to quality check and filter using Fastp [1] .
  • Read mapping to human genome hg19 is performed using BWA MEM with the command “bwa mem -t 8 -M” [2] .
  • Duplicates are removed and local realignment is performed using the GATK bundle [3] .
  • Genotypes (pileup reads) of the D0 and Dx samples on all positions are determined using the mpileup function of Samtools [4] .
  • SNPs are called if the read genotype (s) differ between D0 and Dx samples (see Figure 1A) .
  • the global donor DNA fraction for each transplant recipient at each time point is estimated based on genome-wide SNP genotyping [1, 2] (see Figure 1B) . Consistent with previous findings, donor DNA fraction peaks immediately after transplantation (day 1) and fell quickly (by day 4) .
  • FIG. 1 A) Study design and data analysis flowchart. Blood samples were collected at day 0 (pre-transplant) , day 1, 4, 7, 10, 13 (post-transplant) from recipients of lung transplantation (LTx) . Genomic DNA (gDNA) and cell-free DNA (cfDNA) was extracted from the blood samples and subjected to high coverage whole-genome sequencing. Sequencing data was analysed together with follow-up clinical information. A global donor DNA fraction is estimated for each patient at each time point. Over-representation of graft DNA was examined at SNP-level and region-level. See supplementary Methods for a full description of data processing and analysis. B) Dynamics of global donor DNA fraction during the first two weeks after LTx. Global donor DNA fraction of the three patients at days 1, 4, 7, 10, 13 (post-transplant) were predicted using the genome-wide SNP-based method. Detailed values of global donor DNA fraction for all samples can be found in table 1.
  • Figures 4-6 Circos plots of estimated regional donor DNA fraction on 500kb windows for each recipient at each time point. Time points are arranged from inside to outside -the innermost circle is day 1, and the outermost circle is day 13. The heights of the bars are proportional to the values of the estimated donor DNA fraction. Blank regions indicate lack of informative SNPs at the corresponding window.
  • the circos plot is powered by Bio-oviz circos plot tool: https: //bio. oviz. org/demo-project/analyses/Circos .
  • HLA human leukocyte antigen
  • nucleosome foot-printing of genes of interest were performed as previously described [12, 13] .
  • a nucleosome footprint for a gene is the relative coverage (position depth divided by average depth) at ⁇ 1kb of the transcription start site (TSS) .
  • TSS transcription start site
  • nucleosome footprints [6] of genes within the significant regions pre-and post-transplant are computed. Characteristics of open chromatin are found in some of the genes post-transplant, suggesting active transcriptions (Figure 3B) . Among these likely active genes, some are known to be expressed in lung epithelial during inflammation and transplantation, e.g. DEFB103, DDR1 and MICA [7–9] ; some known to be expressed in both the host and the graft, e.g. HLA-B/C.
  • DEFB103 and DDR1 which are expected to be expressed only in the graft, showed different nucleosome footprints pre-and post-transplant, while genes known to express in both, e.g. HLA-B/C, preserved the shape of the footprints.

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Abstract

L'invention concerne un procédé de détection d'une fraction d'ADNcf donneur dans un échantillon d'un receveur. L'invention concerne également un procédé de détection d'un ou de plusieurs SNP sur-représentés d'ADNcf donneur dans une région identifiée.
PCT/CN2022/140435 2021-12-22 2022-12-20 Procédé de surveillance d'une fraction d'adn donneur Ceased WO2023116717A1 (fr)

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