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What Causes Genomic Instability During Preimplanation Embryo Development? David L

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What Causes Genomic Instability During Preimplanation Embryo Development? David L What Causes Genomic Instability During Preimplanation Embryo Development? David L. Keefe, M.D. SARG International Summit May 28, 2018 Disclosures • Clinical Experts Panel‐Illumina • Scientific Advisory Boards‐ Origio and Cooper Genomics • Research funded by March of Dimes to study effects of telomere attrition on aneuploidy in human embryos Objectives • Describe the pervasive genomic instability that characterizes preimplantation embryo development • Understand how telomeres reset during preimplantation development • Propose an explanation for genomic instability in embryos as a byproduct of telomere rejuvenation during the earliest stages of life I. Global Genomic Instability, Including Aneuploidy, Mosaicism & Copy Number Variants, is Common In Preimplantation Embryos Aneuploidy Contributes to Age‐Related Infertility NYU Data 52% 50% 50.50% 50% 47.70% 35% SART 27% 18% Euploid 9% 2% <35 35‐37 38‐40 41‐42 >42 Harton, Grifo, Munne et al. (2013) Fertil and Steril and unpublished data. What About Mosaicism? • Definition‐ presence of two or more populations of cells with different genotypes in one individual, who has developed from a single fertilized egg.[1] [1] Stern, C. and K. Sekiguti 1931. Bio. Zentr. 51, 194–199. Does Mosaicism Invalidate PGT‐A? • Retrospective study of embryos that had undergone PGS by NGS (@ Reprogenetics) from 2/15‐1/17. • Thawed, expanded, re‐biopsied, repeated NGS using same WGA, Veriseq, MiSeq, but in our own laboratory • Blastocysts w/ diagnosis of mosaic, aneuploid or euploid • Whole Genome Amplification (WGA) by Sureplex • Veriseq DNA library prep • MiSeq sequencing to assess chromosomal copy number • BlueFuse Multi software • Aneuploidy‐ >80% change in chromosome copy number • Mosaicism‐ examined varying thresholds from 20‐80% (per PGDIS); also looked at 30% and 40% thresholds Sachdev et al, ASRM 2017 PGT‐A Is Reliable and Accurate • 129 samples from 32 blastocysts from 17 patients • 18 mosaic, 4 aneuploid and 10 euploid • Concordance for aneuploidy‐ 97% (62/64 samples) • Concordance b/w ICM/TE for aneuploidy‐ 100% • 2 non‐concordant samples between TE biopsies, but both showed either aneuploidy for another chromosome or complex mosaicism with more than 3 chromosomes mosaic. Sachdev et al, ASRM 2017 PGT‐A Is Reliable & Accurate • Mosaic chromosome not identified consistently in repeat biopsy specimens, regardless of diagnostic criteria used to call mosaicism • Concordance for mosaicism between clinical and experimental biopsies not affected by varying diagnostic threshold for mosaicism • Concordance for mosaicism not different between TE and ICM • Number of samples with additional mosaic chromosomes (a chromosome in addition to the chromosome previously diagnosed to be mosaic) significantly varied between the different diagnostic thresholds (p <0.001). • Number of cells included in the biopsy did not affect concordance for mosaicism across all diagnostic threshold levels Sachdev et al, ASRM 2017 Aneuploid Euploid “Euploid” embryo Aneuploid Euploid “Aneuploid” embryo Aneuploid Euploid “Mosaic” embryo Summary and Conclusions‐ Mosaicism & Reliability PGS by NGS • PGS previously thought to provide a binary output‐ normal vs. abnormal • With increased sensitivity associated with aCGH and NGS, PGS now detects more subtle abnormalities, including mosaicism • PGS w/ NGS provides a nuanced output, similar to glucose tolerance test for diagnosis of diabetes mellitus • More studies needed to optimize clinical utility of PGS Copy Number Variants • Definition: a phenomenon in which sections of the genome are repeated and the number of repeats in the genome varies between individuals in the human population.[1] Copy number variation is a type of structural variation: specifically, it is a type of duplication or deletion event that affects a considerable number of base pairs.[2] [1] Mccarroll et al (2007) Nature Gen 39:37‐42 [2]Sharp AJ et al (2005) Am J Hum Gen 77:78‐88 Copy Number Variants Are Common During Preimplantation Embryo Development Why So Much Genomic Instability During Early Embryo Development? • Causes of Mosaicism‐ – Mitotic non‐disjunction, leading to aneuploid cell line – Correction of prior meiotic error • Causes of Copy Number Variants – Anaphase lag‐ is the most common way by which mosaicism arises in the preimplantation embryo.[2] • Anaphase lag‐ delayed movement during anaphase, where one homologous chromosome in meiosis or one chromatid in mitosis fails to connect to the spindle apparatus, or is tardily drawn to its pole and fails to be included in the reforming nucleus. • Chromosome forms a micronucleus in the cytoplasm and is lost from the cell.[1] The lagging chromosome is not incorporated into the nucleus of one of the daughter cells, resulting in one normal daughter cell and one with monosomy.[2] • Anaphase lag can also cause a rescue of the daughter cell if the cell was originally trisomy.[1] [1] Gardner, R.J.M; Sutherland, Grant R. (2004). Chromosome Abnormalities and Genetic Counseling (3rd ed.). NY: Oxford Press. p [2]Jump up^ "Human Molecular Genetics". Archived from the original on June 29, 2007. Anaphase Lag II. Telomeres & Genomic Instability in Preimplantation Embryos Telomere‐ Structure • Tandem repeats (TTAGGG) • 6 proteins in a complex known as Shelterin. • Fold single stranded end of chromosome into a t‐ loop to avoid DNA damage response. • Loss of telomere leads to apoptosis or senescence. Telomere End Fusions Produce Anaphase Lag, Mosaicism & CNVs Maciejowski J Nat Rev (2017)18:175‐86 Telomere Attrition in Mice Phenocopies Reproductive Aging in Women Observation Explained by Telomere Theory Production Line – (Henderson and Edwards, 1968) Decreased Chiasmata- (Hassold et al, 1996) Spindle abnormalities- (Battaglia et al,1997) Low levels mtDNA deletions & ROS - (Keefe et al, 1995) Increased embryo fragmentation & apoptosis (Juriscova et al, 2003) Keefe & Liu, 2009 What About Women? Reproductive Function in Patient w/ Telomeropathy Unaffected Patient (29‐31) DKC (30 years old) Anti‐Mullerian Hormone 1‐30.3 Stimulation Dosage 200 IU 600IU Stimulation Duration 10‐11 18 Oocytes Retrieved 15‐20 7 % Fertilized 90% 40% % of Euploid embryos 50% 14% Robinson et al, unpublished 2007 Telomeres Are Short in Oocytes and Elongate in Embryos‐ Even Parthenotes Telomere Elongation‐ Even in Telomerase Null Mice Telomeres Lengthen in Preimplantation Human Embryos P = .03 N.S. P= .0002 P= .03 Robinson et al, unpublished Telomeres Lengthen in Human Parthenotes Following Activation P = .003 P = .019 N.S. N= 37 N= 21N= 16 N= 32 Robinson et al, unpublished DNA Double Strand Break Repair‐ Rad 50, Bloom, Werner Expressed During Cleavage Stage III. What Causes Alternative Lengthening of Telomeres in Preimplantation Embryos? Flies Elongate Telomeres Via Retrotransposition Retrotransposition • With other, inactive transposable elements, e.g. Alu & Sine, retrotransposons comprise 40% of human genome • Parasites which infected our ancestral genome over billions of years • Line 1‐ Long Interspersed Element (L1)‐ Are active, more recent retrotransposons‐ maintain reverse transcriptase & endonuclease activities. Insert randomly throughout the genome under certain circumstances, e.g. during early embryo development. • Comprises 17% of human genome‐ up to 100 copies active • Held in check by methylation of CpG islands in 5’ promoter region and by histone methylation • During oogenesis & early embryo development cytosines are demethylated to reset the epigenetic clock @ the beginning of life • Cytosine demethylation activates L1 • Activation of L1 causes fetal oocyte attrition in mice Cytosine Demethylation During Gametogenesis & Early Development Do Mammals Mobilize Retrotransposons To Elongate Telomeres During Early Development? Telomere elongation ORF2 expressed in embryos? (Bourroul) inhibited by AZT (Navarro) Telomere RNA (TERRA) Retrotransposon Expressed (Wang) capture (RC) seq to compare TE architecture in euploid vs. aneupoid human embryos; ALT in human persistence of embryos & LINE1 message parthenotes in abnormal (Robinson) blasts? (Maxwell) Methylome clock advanced in individual human oocytes? (Wang, Charmani) TERRA Biogenesis in Mammals S. Feuerhahn et al. / FEBS Letters 584 (2010) 3812–3818 TERRA Activated in Cleavage Mouse Embryos A TERRA TRF1 DAPI Merge B Early 2cell 7.5 (n=6) (G1) Late 20 (n=6) 2cell (S/G2) >50 (n=12) 4cell TERRA foci per cell Line1 Copy Number Increases During Early Development Summary & Conclusions • Genomic instability is common in preimplantation embryos • Mitotic as well as meiotic errors‐ mosaicism, copy number variants • Telomeres are essential for genomic stability • DNA recombination‐based telomere elongation, which resembles retrotransposon activation, may contribute to global genomic instability during preimplantation embryo development Acknowledgements Laboratory for Human Reproduction David L. Keefe Fang ‘Helen’ Wang Ricardo Pimentel (Univ. Fed. De Goias) Libing Wang (UCLA) Lan Wang (Tongji Med. College) Elisa Atamian (Boston Univ.) Jason Kofinas (Kofinas Ferility) Dani Antunes (NYU FC) Molly Kumar (University of Sydney) Lin Liu (Univ. Nankai) Nidhee Sachdev Paula Navarro (USP‐RP, Brazil) NYU Fertility Center New York Stem Cell Foundation Funding Yael Kramer Deter Egli March of Dimes David McCulloh Stanley Kaplan Research Jamie Grifo & Endowment Embryology Staff.
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