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Johnson D.S.,Gene Security Network | Gemelos G.,Gene Security Network | Baner J.,Gene Security Network | Baner J.,Stanford University | And 12 more authors.
Human Reproduction | Year: 2010

Background Preimplantation genetic screening (PGS) has been used in an attempt to determine embryonic aneuploidy. Techniques that use new molecular Method s to determine the karyotype of an embryo are expanding the scope of PGS.Method SWe introduce a new Method for PGS, termed 'parental support', which leverages microarray measurements from parental DNA to 'clean' single-cell microarray measurements on embryonic cells and explicitly computes confidence in each copy number call. The Method distinguishes mitotic and meiotic copy errors and determines parental source of aneuploidy.Result SValidation with 459 single cells of known karyotype indicated that per-cell false-positive and false-negative rates are roughly equivalent to the 'gold standard' metaphase karyotype. The majority of the cells were run in parallel with a clinical commercial PGS service. Computed confidences were conservative and roughly concordant with accuracy. To examine ploidy in human embryos, the Method was then applied to 26 disaggregated, cryopreserved, cleavage-stage embryos for a total of 134 single blastomeres. Only 23.1 of the embryos were euploid, though 46.2 of embryos were mosaic euploid. Mosaicism affected 57.7 of the embryos. Counts of mitotic and meiotic errors were roughly equivalent. Maternal meiotic trisomy predominated over paternal trisomy, and maternal meiotic trisomies were negatively predictive of mosaic euploid embryos.Conclusion SWe have performed a major preclinical validation of a new Method for PGS and found that the technology performs approximately as well as a metaphase karyotype. We also directly measured the mechanism of aneuploidy in cleavage-stage human embryos and found high rates and distinct patterns of mitotic and meiotic aneuploidy.


Johnson D.S.,Gene Security Network | Cinnioglu C.,Gene Security Network | Ross R.,La Jolla IVF | Filby A.,Northern California Fertility Medical Center | And 6 more authors.
Molecular Human Reproduction | Year: 2010

Aneuploidy has been well-documented in blastocyst embryos, but prior studies have been limited in scale and/or lack mechanistic data. We previously reported preclinical validation of microarray 24-chromosome preimplantation genetic screening in a 24-h protocol. The method diagnoses chromosome copy number, structural chromosome aberrations, ntal source of aneuploidy and distinguishes certain meiotic from mitotic errors. In this study, our objective was to examine aneuploidy in human blastocysts and determine correspondence of karyotypes between trophectoderm (TE) and inner cell mass (ICM). We disaggregated 51 blastocysts from 17 couples into ICM and one or two TE fractions. The average maternal age was 31. Next, we ran 24-chromosome microarray molecular karyotyping on all of the samples, and then performed a retrospective analysis of the data. The average per-chromosome confidence was 99.95%. Approximately 80% of blastocysts were euploid. The majority of aneuploid embryos were simple aneuploid, i.e. one or two whole-chromosome imbalances. Structural chromosome aberrations, which are common in cleavage stage embryos, occurred in only three blastocysts (5.8%). All TE biopsies derived from the same embryos were concordant. Forty-nine of 51 (96.1%) ICM samples were concordant with TE biopsies derived from the same embryos. Discordance between TE and ICM occurred only in the two embryos with structural chromosome aberration. We conclude that TE karyotype is an excellent predictor of ICM karyotype. Discordance between TE and ICM occurred only in embryos with structural chromosome aberrations. © The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.


Landolin J.M.,Lawrence Berkeley National Laboratory | Johnson D.S.,Stanford University | Johnson D.S.,Gene Security Network | Trinklein N.D.,SwitchGear Genomics | And 7 more authors.
Genome Research | Year: 2010

Promoters are important regulatory elements that contain the necessary sequence features for cells to initiate transcription. To functionally characterize a large set of human promoters, we measured the transcriptional activities of 4575 putative promoters across eight cell lines using transient transfection reporter assays. In parallel, we measured gene expression in the same cell lines and observed a significant correlation between promoter activity and endogenous gene expression (r = 0.43). As transient transfection assays directly measure the promoting effect of a defined fragment of DNA sequence, decoupled from epigenetic, chromatin, or long-range regulatory effects, we sought to predict whether a promoter was active using sequence features alone. CG dinucleotide content was highly predictive of ubiquitous promoter activity, necessitating the separation of promoters into two groups: high CG promoters, mostly ubiquitously active, and low CG promoters, mostly cell line-specific. Computational models trained on the binding potential of transcriptional factor (TF) binding motifs could predict promoter activities in both high and low CG groups: average area under the receiver operating characteristic curve (AUC) of the models was 91% and exceeded the AUC of CG content by an average of 23%. Known relationships, for example, between HNF4A and hepatocytes, were recapitulated in the corresponding cell lines, in this case the liver-derived cell line HepG2. Half of the associations between tissue-specific TFs and cell line-specific promoters were new. Our study underscores the importance of collecting functional information from complementary assays and conditions to understand biology in a systematic framework. © 2010 by Cold Spring Harbor Laboratory Press.


Rabinowitz M.,Gene Security Network | Rabinowitz M.,Stanford University | Ryan A.,Gene Security Network | Gemelos G.,Gene Security Network | And 9 more authors.
Fertility and Sterility | Year: 2012

Objective: To characterize chromosomal error types and parental origin of aneuploidy in cleavage-stage embryos using an informatics-based technique that enables the elucidation of aneuploidy-causing mechanisms. Design: Analysis of blastomeres biopsied from cleavage-stage embryos for preimplantation genetic screening during IVF. Setting: Laboratory. Patient(s): Couples undergoing IVF treatment. Intervention(s): Two hundred seventy-four blastomeres were subjected to array-based genotyping and informatics-based techniques to characterize chromosomal error types and parental origin of aneuploidy across all 24 chromosomes. Main Outcome Measure(s): Chromosomal error types (monosomy vs. trisomy; mitotic vs. meiotic) and parental origin (maternal vs. paternal). Result(s): The rate of maternal meiotic trisomy rose significantly with age, whereas other types of trisomy showed no correlation with age. Trisomies were mostly maternal in origin, whereas paternal and maternal monosomies were roughly equal in frequency. No examples of paternal meiotic trisomy were observed. Segmental error rates were found to be independent of maternal age. Conclusion(s): All types of aneuploidy that rose with increasing maternal age can be attributed to disjunction errors during meiosis of the oocyte. Chromosome gains were predominantly maternal in origin and occurred during meiosis, whereas chromosome losses were not biased in terms of parental origin of the chromosome. The ability to determine the parental origin for each chromosome, as well as being able to detect whether multiple homologs from a single parent were present, allowed greater insights into the origin of aneuploidy. © 2012 American Society for Reproductive Medicine, Published by Elsevier Inc.


PubMed | Gene Security Network
Type: Comparative Study | Journal: Human reproduction (Oxford, England) | Year: 2010

Preimplantation genetic screening (PGS) has been used in an attempt to determine embryonic aneuploidy. Techniques that use new molecular methods to determine the karyotype of an embryo are expanding the scope of PGS.We introduce a new method for PGS, termed parental support, which leverages microarray measurements from parental DNA to clean single-cell microarray measurements on embryonic cells and explicitly computes confidence in each copy number call. The method distinguishes mitotic and meiotic copy errors and determines parental source of aneuploidy.Validation with 459 single cells of known karyotype indicated that per-cell false-positive and false-negative rates are roughly equivalent to the gold standard metaphase karyotype. The majority of the cells were run in parallel with a clinical commercial PGS service. Computed confidences were conservative and roughly concordant with accuracy. To examine ploidy in human embryos, the method was then applied to 26 disaggregated, cryopreserved, cleavage-stage embryos for a total of 134 single blastomeres. Only 23.1% of the embryos were euploid, though 46.2% of embryos were mosaic euploid. Mosaicism affected 57.7% of the embryos. Counts of mitotic and meiotic errors were roughly equivalent. Maternal meiotic trisomy predominated over paternal trisomy, and maternal meiotic trisomies were negatively predictive of mosaic euploid embryos.We have performed a major preclinical validation of a new method for PGS and found that the technology performs approximately as well as a metaphase karyotype. We also directly measured the mechanism of aneuploidy in cleavage-stage human embryos and found high rates and distinct patterns of mitotic and meiotic aneuploidy.


PubMed | Gene Security Network
Type: Journal Article | Journal: Fertility and sterility | Year: 2012

To characterize chromosomal error types and parental origin of aneuploidy in cleavage-stage embryos using an informatics-based technique that enables the elucidation of aneuploidy-causing mechanisms.Analysis of blastomeres biopsied from cleavage-stage embryos for preimplantation genetic screening during IVF.Laboratory.Couples undergoing IVF treatment.Two hundred seventy-four blastomeres were subjected to array-based genotyping and informatics-based techniques to characterize chromosomal error types and parental origin of aneuploidy across all 24 chromosomes.Chromosomal error types (monosomy vs. trisomy; mitotic vs. meiotic) and parental origin (maternal vs. paternal).The rate of maternal meiotic trisomy rose significantly with age, whereas other types of trisomy showed no correlation with age. Trisomies were mostly maternal in origin, whereas paternal and maternal monosomies were roughly equal in frequency. No examples of paternal meiotic trisomy were observed. Segmental error rates were found to be independent of maternal age.All types of aneuploidy that rose with increasing maternal age can be attributed to disjunction errors during meiosis of the oocyte. Chromosome gains were predominantly maternal in origin and occurred during meiosis, whereas chromosome losses were not biased in terms of parental origin of the chromosome. The ability to determine the parental origin for each chromosome, as well as being able to detect whether multiple homologs from a single parent were present, allowed greater insights into the origin of aneuploidy.


There is disclosed a method for determining the number of copies of a chromosome, or segment of a chromosome, in the genome of a target individual. The method comprises (i) creating a set of one or more hypotheses about the number of instances of the given chromosome or chromosomes segment present in the genome of the target individual, (ii) obtaining genetic data for some or all of the possible alleles at a plurality of loci on the given chromosome or chromosome segment of the target individual, (iii) obtaining genetic data of one or more related individuals for some or all of the possible alleles at a plurality of loci on the given chromosome or chromosome segment, (iv) determining the relative probability of each of the hypotheses given the obtained genetic data of the individuals, and (v) using the relative probabilities associated with each hypothesis to determine the likely number of instances of the chromosome or the segment of a chromosome in the actual genetic material of the target individual. A data processing system and a computer-readable medium are also disclosed.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.60M | Year: 2011

DESCRIPTION (provided by applicant): During the course of a pregnancy, physicians and patients desire as much information as possible regarding the health of the fetus. For both emotional and medical reasons, this information is sought as early in term aspossible, and with the fewest possible risks to both mother and child. Although the widely used first trimester chorionic villus sampling (CVS) and second trimester amniocentesis are relatively safe, both procedures are not without negligible risks. In efforts to avoid these risks altogether, researchers have turned toward isolating circulating fetal nucleated red blood cells (FNRBCs) from maternal blood as an alternative, non- invasive source of fetal tissue. Despite the development of FNRBC enrichment methods, there has been limited success with their coupling to subsequent aneuploidy screening and several challenges still must be overcome such as ability to test single fetal cells for 24-chromosome aneuploidy, confirm the isolated cell's origin (fetal versus maternal) and simultaneously screen for diseases caused by single nucleotide variants or micro in/dels. Our innovative Parental SupportTM technology provides a solution to all of these challenges and the development of a first trimester non-invasiveprenatal diagnostic test is the ultimate goal of this grant application. In Phase I, we first plan to optimize single cell lysis and whole genome amplification protocols specifically for antibody-stained FNRBCs.. Protocol optimization for single cell analysis falls within the core competencies of GSN as we have previously successfully commercialized an innovative single cell molecular karyotyping protocol to enable genetic analysis of single blastomeres within 24 hours. We will then systematically evaluatewhich combination of existing FNRBC enrichment methods provides maximum yield and purity suitable for subsequent Parental Support -based genetic analysis using predefined mixtures of fetal and adult blood. The main objective of Phase II will be to transition from the predefined blood mixtures of fetal and adult blood to actual maternal blood samples. We will first conduct a pilot study to determine which of the best FNRBC isolation method(s) identified in Phase I should become the lead method. Using this lead method, we will then conduct a larger study to evaluate concordance between aneuploidy diagnosis by Parental SupportTM and karyotyping by amniocentesis or chorionic villus sampling. If successful, we expect that the completion of these Aims would have amajor impact on the field of prenatal diagnosis, improve the lives of millions of couples and children worldwide, and bring non-invasive diagnosis to the mainstream of prenatal medicine. PUBLIC HEALTH RELEVANCE: In the absence of prenatal diagnosis, up to 1 in 50 babies have serious physical or mental handicaps, up to 1 in 30 babies have some form of congenital malformation, and up to 1 in 200 have a phenotypically significant chromosome abnormality Although these abnormalities can be diagnosed withtechniques such as amniocentesis or chorionic villus sampling, both procedures carry an increased risk of harm to both the mother and fetus. Our innovative technology has the potential to evaluate the health of an unborn child by simply analyzing the mother's blood, thereby minimizing the risks of the procedure and expanding prenatal screening to the general population.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 200.83K | Year: 2010

DESCRIPTION (provided by applicant): During the course of a pregnancy, physicians and patients desire as much information as possible regarding the health of the fetus. For both emotional and medical reasons, this information is sought as early in term as possible, and with the fewest possible risks to both mother and child. Although the widely used first trimester chorionic villus sampling (CVS) and second trimester amniocentesis are relatively safe, both procedures are not without negligible risks. In efforts to avoid these risks altogether, researchers have turned toward isolating circulating fetal nucleated red blood cells (FNRBCs) from maternal blood as an alternative, non- invasive source of fetal tissue. Despite the development of FNRBC enrichment methods, there has been limited success with their coupling to subsequent aneuploidy screening and several challenges still must be overcome such as ability to test single fetal cells for 24-chromosome aneuploidy, confirm the isolated cell's origin (fetal versus maternal) and simultaneously screen for diseases caused by single nucleotide variants or micro in/dels. Our innovative Parental SupportTM technology provides a solution to all of these challenges and the development of a first trimester non-invasive prenatal diagnostic test is the ultimate goal of this grant application. In Phase I, we first plan to optimize single cell lysis and whole genome amplification protocols specifically for antibody-stained FNRBCs.. Protocol optimization for single cell analysis falls within the core competencies of GSN as we have previously successfully commercialized an innovative single cell molecular karyotyping protocol to enable genetic analysis of single blastomeres within 24 hours. We will then systematically evaluate which combination of existing FNRBC enrichment methods provides maximum yield and purity suitable for subsequent Parental Support -based genetic analysis using predefined mixtures of fetal and adult blood. The main objective of Phase II will be to transition from the predefined blood mixtures of fetal and adult blood to actual maternal blood samples. We will first conduct a pilot study to determine which of the best FNRBC isolation method(s) identified in Phase I should become the lead method. Using this lead method, we will then conduct a larger study to evaluate concordance between aneuploidy diagnosis by Parental SupportTM and karyotyping by amniocentesis or chorionic villus sampling. If successful, we expect that the completion of these Aims would have a major impact on the field of prenatal diagnosis, improve the lives of millions of couples and children worldwide, and bring non-invasive diagnosis to the mainstream of prenatal medicine. PUBLIC HEALTH RELEVANCE: In the absence of prenatal diagnosis, up to 1 in 50 babies have serious physical or mental handicaps, up to 1 in 30 babies have some form of congenital malformation, and up to 1 in 200 have a phenotypically significant chromosome abnormality Although these abnormalities can be diagnosed with techniques such as amniocentesis or chorionic villus sampling, both procedures carry an increased risk of harm to both the mother and fetus. Our innovative technology has the potential to evaluate the health of an unborn child by simply analyzing the mother's blood, thereby minimizing the risks of the procedure and expanding prenatal screening to the general population.


PubMed | Gene Security Network
Type: Comparative Study | Journal: Molecular human reproduction | Year: 2010

Aneuploidy has been well-documented in blastocyst embryos, but prior studies have been limited in scale and/or lack mechanistic data. We previously reported preclinical validation of microarray 24-chromosome preimplantation genetic screening in a 24-h protocol. The method diagnoses chromosome copy number, structural chromosome aberrations, parental source of aneuploidy and distinguishes certain meiotic from mitotic errors. In this study, our objective was to examine aneuploidy in human blastocysts and determine correspondence of karyotypes between trophectoderm (TE) and inner cell mass (ICM). We disaggregated 51 blastocysts from 17 couples into ICM and one or two TE fractions. The average maternal age was 31. Next, we ran 24-chromosome microarray molecular karyotyping on all of the samples, and then performed a retrospective analysis of the data. The average per-chromosome confidence was 99.95%. Approximately 80% of blastocysts were euploid. The majority of aneuploid embryos were simple aneuploid, i.e. one or two whole-chromosome imbalances. Structural chromosome aberrations, which are common in cleavage stage embryos, occurred in only three blastocysts (5.8%). All TE biopsies derived from the same embryos were concordant. Forty-nine of 51 (96.1%) ICM samples were concordant with TE biopsies derived from the same embryos. Discordance between TE and ICM occurred only in the two embryos with structural chromosome aberration. We conclude that TE karyotype is an excellent predictor of ICM karyotype. Discordance between TE and ICM occurred only in embryos with structural chromosome aberrations.

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