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Preimplantation Genetic Diagnosis (PGD)

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Preimplantation Genetic Diagnosis

(PGD)

AS3323/5621
Lecture 7
Sept 19, 2017

1

Risks of Fetal Loss

2

A total of 634,272 women and 1,221,546 pregnancy outcomes in Denmark from 1978 to 1992.
Anderson et al. BMJ, 2000,320:1708-1712.

Meiotic Non-disjunction:% Trisomy?

3

Aneuploidy

P

centromere

Q

Abnormal number of chromosomes in a cell.

4

Aneuploidy: the most common cause for early pregnancy failure

Prevalence of oocyte and embryo aneuploidy increases with maternal age

Also increase in chromosomally normal couples with recurrent early pregnancy loss or repeated failed IVF cycles despite the transfer of high-quality embryos (based on morphology).

http://www.asrm.org/uploadedFiles/ASRM_Content/News_and_Publications/Practice_Guidelines/Commi ttee_Opinions/Preimplantation_genetic_testing(1).pdf
5

“Common” Types of Trisomy

Trisomy 21 – Down’s Syndrome

karyotype 47, XX +21 or 47, XY + 21 frequency ~ 1/600 births

Trisomy 18 – Edward’s Syndrome

karyotype 47, XX + 18 or 47, XY + 18 Frequency ~ 1 in 10,000 births

Sex chromosome trisomies
47, XXY (Klinefelter Syndrome), 1/1,000 males 47, XXX (super females), many un-diagnosed Polysomy X e.g., XXXX

Trisomies of other chromosomes partial, mosaic and rare incompatible with life

6

Monosomy

• No other live births of full monosomy

– abortion

• Cancer cells (lymphocytes) • Only partial monosomies in live births

• Turner’s Syndrome: XO females

7

Why does aneuploidy lead to fetal

death?

Gene dosage

• Intolerance to deviations from diploid • Extra or missing chromosomes causes developmental defects and/or death (monosomy, trisomy)

• Sex chromosomes exception
• On the surface

8

Central Dogma of Gene Expression

9

http://cnx.org/contents/Z7qBU2RZ@5/The-Central-Dogma-and-Basic-Tr

Gene copy number vs. Dosage

Two copies, two doses

One copy,

two doses

10

Gene Dosage Problem ?

XX

XY

Gene Dosage Problem ?

AA

X-

XY

Basic Steps of IVF-PGD

1. Regular IVF (can be for fertile couples) 2. Embryo biopsy: day 3 (what cell stage?) 3. PGD by cellular/molecular analyses 4. Determine which embryos to transfer

  • 13
  • 14

Potency of Early Cleavage Stage

Blastomeres (animal model)

The world's first identical quadruplet bulls produced from a single four-cell embryo by in vitro

fertilization were born at the University of Guelph in the fall of 1992.

Johnson et al. Veterinary Record 1995, 137:15-16.

15

PGD Molecular Techniques

– fluorescence in situ hybridization (FISH)

– comparative genomic hybridization

(CGH)
– array CGH (aCGH) – digital polymerase chain reaction (dPCR) – single-nucleotide polymorphism (SNP) array
– real-time quantitative PCR (qPCR) – next-generation sequencing (NGS).

ASRM Practice Committee Brief Communication on Pre-Implantation Genetic Screening

16

for Aneuploidy: a committee opinion, March 2016

Single Cell Genetic Analysis

Pre-implantation Genetic Diagnosis is a technique based on single cell genetic analysis at the chromosomal or nucleotide level.

17

Major Categories Of PGD

1. Chromosome number: Aneuploidy

2. Chromosome structure

– Translocation, deletion, – duplication, inversion

3. Single gene disorder 4. X-linked disorders 5. Social sexing

18

PGD Molecular Methods

• FISH: Fluorescence In Situ
Hybridization

• PCR: Polymerase Chain Reaction
(see PGD example #2)

19

Fluorescence In Situ Hybridization

http://www.youtube.com/watch?v=nm8Ai1CI9Is

20

AS 5621:needs to know procedure

FISH Probes

Centromeric probes

21

Locus specific / telomeric probes
Whole chromosome painting probes

Interphase Chromatin, Metaphase

Chromosomes

22

Chromatin are relatively condensed in blastomeres

Aneuploidy Analysis with FISH

Nucleus

fixation

Denaturation

of probes/nucleus

Addition of

probes

Biopsy

13 = red

16 = aqua

18 = blue

21 = green

22 = yellow

Wash to eliminate non-specific hybridization
Visualization with proper filters
Hybridization (4 hrs / overnight)

Wash off probes

  • 22
  • 13

Y

Re-hybridize with

X = yellow

Y = aqua

15 = green

17 = orange

  • 16
  • 17

18

15
X
21

23

9 probes: X, Y, 13, 15, 16, 17, 18, 21, 22

Aneuploidy Analysis

(Screening of 9 Chromosomes)

18

13

15
21

22

15

16

X
22

18

17
17

16

13

21

Y

1st FISH

2nd FISH
X (yellow) Y (aqua)
15 (green) 17 (orange)

13 (red) 16 (aqua) 18 (blue) 21 (green) 22 (gold)

XY, 13/13, 15/15, 16/16, 17/17, 18/18, 21/21, 22/22

24

Problem of this approach?

Chromosome Numerical Evaluation
Currently the Major Indication for PGD

•Avoid live-births with chromosomal disorders (eg, Down, Turner (XO), etc)

•Reduce risks of miscarriages

•Increase pregnancy rates?

•Not with FISH alone •With new methods, yes !!!

•Prior believe: common aneuploidy (13, 16, 18, 21, 22, X and Y), but later found aneuploidy can happen all chromosomes.

25

Trisomy 21 due to abnormal cell division

26

Down’s Syndrome

• Impaired learning and physical growth, and a recognizable facial appearance usually identified at birth

• Trisomy 21, accounts for 95% of Down’s Syndrome

• Most, 88% 2 copies of maternal 21

• Down’s syndrome is caused in a minority (5% or less)

of cases by a Robertsonian translocation of about a third of chromosome 21 onto chromosome 14.

27

Maternal Age and Trisomy 21

Eve fertile

women should

be careful when reproducing at an older age

Michelle Duggar (50): 17 births (2 sets of twins) in 21 years, ~15 month/birth; last a premie born in 2009 at 42; miscarriage in 2011 at 44.

28

Chromosome Structural Analysis

• Balanced translocation

• Deletion • Inversion

29

Chromosome Translocations

• Robertsonian translocations • Reciprocal translocations

• When balanced, carriers are normal

30

Recommended publications
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  • MET-Overexpressing Myxofibrosarcoma Frequently

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    Leukemia (2003) 17, 2225–2229 & 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu Translocations t(11;18)(q21;q21) and t(14;18)(q32;q21) are the main chromosomal abnormalities involving MLT/MALT1 in MALT lymphomas EM Murga Penas1, K Hinz1,KRo¨ser2, C Copie-Bergman3, I Wlodarska4, P Marynen4, A Hagemeijer4, P Gaulard3,TLo¨ning2, DK Hossfeld1 and J Dierlamm1 1Department of Medicine, Hamburg, Germany; 2Department of Pathology, University Hospital Hamburg-Eppendorf, Hamburg, Germany; 3Department of Pathology, Centre Hospitalier Universitaire, Henri Mondor, Cre´teil, France; and 4Center for Human Genetics and Flanders Interuniversity, Institute for Biotechnology, University of Leuven, Leuven, Belgium The recently discovered MLT/MALT1 gene is fused with the not respond to Helicobacter pylori eradication therapy and API2 gene in the t(11;18)(q21;q21), which characterizes about H. pylori-negative gastric MALT lymphomas with a rather one-third of MALT lymphomas. In order to screen for variant 15,16 translocations and amplifications of MLT/MALT1, we have aggressive clinical course. developed a novel, undirected two-color interphase fluores- Recently, the t(14;18)(q32;q21) involving the MLT/MALT1 cence in situ hybridization (FISH) assay with two PAC clones and IGH genes has been identified as a new frequent flanking MLT/MALT1. This assay was applied to 108 marginal chromosomal translocation in MALT lymphomas.17,18 The zone B-cell lymphomas (MZBCLs), including 72 extranodal t(14;18) characterizes MALT lymphomas in locations, such as MALT lymphomas, 17 nodal, and 19 splenic MZBCL. In 19 MALT liver, skin, parotid gland, and ocular adnexa that rarely harbor lymphomas (26%), but in none of the nodal or splenic MZBCL, 18 separated hybridization signals of the MLT/MALT1 flanking the t(11;18)(q21;q21).
  • Mosaic Chromosomal Aberrations in Synovial Fibroblasts of Patients With

    Mosaic Chromosomal Aberrations in Synovial Fibroblasts of Patients With

    Available online http://arthritis-research.com/content/3/5/319 Research article Mosaic chromosomal aberrations in synovial fibroblasts of patients with rheumatoid arthritis, osteoarthritis, and other inflammatory joint diseases commentary Raimund W Kinne*, Thomas Liehr†, Volkmar Beensen†, Elke Kunisch*, Thomas Zimmermann*, Heidrun Holland‡, Robert Pfeiffer§, Hans-Detlev Stahl§, Wolfgang Lungershausen¶, Gert Hein††, Andreas Roth‡‡, Frank Emmrich§, Uwe Claussen† and Ursula G Froster‡ *Experimental Rheumatology Unit, Friedrich Schiller University Jena, Jena, Germany †Institute of Human Genetics and Anthropology, Friedrich Schiller University Jena, Jena, Germany ‡Institute of Human Genetics, University of Leipzig, Leipzig, Germany §Institute of Clinical Immunology and Transfusion Medicine, University of Leipzig, Leipzig, Germany ¶Department of Traumatology, Friedrich Schiller University Jena, Jena, Germany ††Clinic of Internal Medicine IV, Friedrich Schiller University Jena, Jena, Germany ‡‡Clinic of Orthopedics, Friedrich Schiller University Jena, Jena, Germany Correspondence: Raimund W Kinne, Experimental Rheumatology Unit, Friedrich Schiller University Jena, Winzerlaer Str. 10, D-07745 Jena, Germany; Tel. +49 3641 65 71 50; fax: +49 3641 65 71 52; e-mail: [email protected] review Received: 19 April 2001 Arthritis Res 2001, 3:319–330 Revisions requested: 31 May 2001 This article may contain supplementary data which can only be found Revisions received: 12 June 2001 online at http://arthritis-research.com/content/3/5/319 Accepted: 22 June 2001 © 2001 Kinne et al, licensee BioMed Central Ltd Published: 3 August 2001 (Print ISSN 1465-9905; Online ISSN 1465-9913) Abstract Chromosomal aberrations were comparatively assessed in nuclei extracted from synovial tissue, primary-culture (P-0) synovial cells, and early-passage synovial fibroblasts (SFB; 98% enrichment; P-1, P-4 [passage 1, passage 4]) from patients with rheumatoid arthritis (RA; n = 21), osteoarthritis (OA; reports n = 24), and other rheumatic diseases.
  • An In-Depth Analysis of the HER2 Amplicon and Chromosome 17

    An In-Depth Analysis of the HER2 Amplicon and Chromosome 17

    Rondón-Lagos et al. BMC Cancer 2014, 14:922 http://www.biomedcentral.com/1471-2407/14/922 RESEARCH ARTICLE Open Access Unraveling the chromosome 17 patterns of FISH in interphase nuclei: an in-depth analysis of the HER2 amplicon and chromosome 17 centromere by karyotyping, FISH and M-FISH in breast cancer cells Milena Rondón-Lagos1,4, Ludovica Verdun Di Cantogno2, Nelson Rangel1,4, Teresa Mele3, Sandra R Ramírez-Clavijo4, Giorgio Scagliotti3, Caterina Marchiò1,2*† and Anna Sapino1,2*† Abstract Background: In diagnostic pathology, HER2 status is determined in interphase nuclei by fluorescence in situ hybridization (FISH) with probes for the HER2 gene and for the chromosome 17 centromere (CEP17). The latter probe is used as a surrogate for chromosome 17 copies, however chromosome 17 (Chr17) is frequently rearranged. The frequency and type of specific structural Chr17 alterations in breast cancer have been studied by using comparative genomic hybridization and spectral karyotyping, but not fully detailed. Actually, balanced chromosome rearrangements (e.g. translocations or inversions) and low frequency mosaicisms are assessable on metaphases using G-banding karyotype and multicolor FISH (M-FISH) only. Methods: We sought to elucidate the CEP17 and HER2 FISH patterns of interphase nuclei by evaluating Chr17 rearrangements in metaphases of 9 breast cancer cell lines and a primary culture from a triple negative breast carcinoma by using G-banding, FISH and M-FISH. Results: Thirty-nine rearranged chromosomes containing a portion of Chr17 were observed. Chromosomes 8 and 11 were the most frequent partners of Chr17 translocations. The lowest frequency of Chr17 abnormalities was observed in the HER2-negative cell lines, while the highest was observed in the HER2-positive SKBR3 cells.