κλινικές...

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) ) , DNA ABCDEFGHIJKLMNOPQRSTUVWXYZabcdefghijklmnopqrstuvwxyzABCDEFGhijklmnoPQRSTUVWXYZabcdefgHIJKLMNOpqrstuvwxyz

( ), 50 . ( 16 24 ), 70 . .

Using semen flow cytometry to evaluate association of ploidy status and chromatin condensation ofspermatozoa with conventional semen parameters: Clinical application in intrauterine inseminationLeandros Lazaros, Ph.D.,a Apostolos Kaponis, M.D.,a Georgios Vartholomatos, Ph.D.,b Elissavet Hatzi, Ph.D.,aStefania Botsari, M.D.,a Nikolaos Plachouras, M.D.,a Georgios Makrydimas, M.D.,aKonstantinos Zikopoulos, M.D.,a Nikolaos Sofikitis, M.D.,c and Ioannis Georgiou, Ph.D.aFertility and Sterility 2010 23- < 23 < 23+ < 46 < 92

(allelic vs non allelic) (inter chromosomal, intrachromosomal, intrachromatid) ( ) DNA (DSBs)

RNA cDNA

1 2

Copy & Paste

DNA DNA, , RNA>cDNA

5

2.87%8.29%20.42%13.29% 0.15% 0.40% 45%

6

1. - 2. 5.

4. 3. (ALU exonization)

7

= :

marker chromosomes

Emanuel and Shaikh, Segmental duplications: an expanding role in genomic instability and disease. Nature Reviews Genetics, Volume 2, October 2001, 791-800.

Segmental duplications & Copy number variations

DNA

FoSTeS (fork stalling and template switching) . MMBIR (microhomology-mediated break-induced replication) . FoSTeS/MMBIR .

aCGH

DeletionDuplicationCopy number variations (CNV)

aCGH

(.. )

aCGH ( ) (CNV) CNV:1. 2. , , 3. (CNVs)

Blake et al.

LINE-1

FoSTeS, MMBIR and NAHR at the human proximal Xp region and the mechanisms of human Xq isochromosome formation.KoumbarisG, et al Hum Mol Genet. 2011

16

(aCGH) aCGH aCGH aCGH

Aneuploidy in the human cleavage stage embryo 60% 3 1 20-25%

Mantzouratou & Delhanty 2011 Review

Polar body analysis by array CGH accurately predicts aneuploidies of maternal meiotic origin in cleavage stage embryos of women of advanced maternal age

Sixty-nine (93%) aneuploidies were associated with copy number changes in the polar bodies and 98.5% of these had been predicted to be aneuploid. Also, 95% balanced combinations of chromatid gain/loss in PB1/PB2 accurately predicted normal copy number in the corresponding embryos. Most of these involved copy number changes that were smaller than would be expected for whole chromosome or chromatid imbalance and occurred significantly more often in PB1 than PB2 (P < 0.0005).

Christopikou et al Hum Repr 2013

Microarray analysis reveals abnormal chromosomal complements in over 70% of 14 normally developing human Embryos

main results and the role of chance: The good-quality embryos exhibited high rates of aneuploidy, 10 of 14 (71.4%) of the embryos being mosaic. While none of the embryos had the same aneuploidy pattern in all cells, 4 of 14 (28.6%) were uniformly diploid.Of the 70 analysed blastomeres, 55.7% were diploid and 44.3% had chromosomal abnormalities, while 29% of the abnormal cells carried structural aberrations.Mertzanidou et al Hum Repr 2012

aCGH :1. 2. 3. (Variant on unknown significance VOUS) . .

CNV :

CNV CNV CNV / (controls) , , ...

DELETION of approximately 1.4Mb in size on the long arm of chromosome 17 (q-arm) at chromosomal band 17q12 (location: 34,823,154-36,248,888). ( )The deletion consists of two OMIM disease genes namely Acetyl-Coa Carboxylase-Alpha (ACACA, OMIM#200350) and the Hepatocyte nuclear factor-1-beta (HNF1B, OMIM#189907) ref 4. The deletion is most probably related to the reason for referral and is consistent with the phenotype. (Mitsioni et al 2014) 1

DUPLICATION of approximately 0.2Mb in size on the long arm of chromosome 5 (q-arm) at chromosomal band 15q23.3 (location: 129,483,166-129,703,662). ( )The duplication partially disrupts one Refseq gene namely Chondroitin Sulfate Synthase 3 (CHY3) which is most probably unrelated to the reason for referral (Mitsioni et al 2014).The change is a VOUS with unknown significance 2

RT-PCR

3

aCGH aCGH : .. ()

aCGH in PND , : VOUS

Additional information from array comparative genomic hybridization technology over conventional karyotypingin prenatal diagnosis: a systematic review and meta-analysisS. C. HILLMAN*, S. PRETLOVE, A. COOMARASAMY*, D. J. McMULLAN, E. V. DAVISON,E. R. MAHER* and M. D. KILBY*

All CNVs pathogenic, of unknown significance or benign included Ultrasound Obstet Gynecol 2011; 37: 614

Additional information from array comparative genomic hybridization technology over conventional karyotypingin prenatal diagnosis: a systematic review and meta-analysisS. C. HILLMAN*, S. PRETLOVE, A. COOMARASAMY*, D. J. McMULLAN, E. V. DAVISON,E. R. MAHER* and M. D. KILBY*

IF ONLY CNVs which are pathogenic or of unknown significance are included advantage drops by 50%Ultrasound Obstet Gynecol 2011

Overall detection rate 5.3%5.5% (140/2533) with significant findings although normal karyotype71% of significant CNVs < 10kb (unlikely detection with karyotyping)Turnaround time mean 7.5 days (median 6 days)Frequency of VOUS 4.2%, de novo 0.4%

The largest study so far N=5003 from one lab Prenat Diagn. 2012 Oct;32(10):976-85

aCGH PNDEthical Considerations Aitomaki 2012Before prenatal CMA patients need counselling forPossibility of identifying VOUSPossibility of incidental findings

Patients need to decide what information they want to receive, particularly concerning incidental findings

In conclusionWe should provide our patients with the benefits or modern genetic technology in prenatal testing

Dr. Diana Wellesley M.D., FRCP, Head, Prenatal Genetics, WessexClinical Genetics Service, Princess Anne Hospital (United Kingdom) Prenatal Arrays in the UK - Proposals for a National Approach 2014

The diagnostic superiority of aCGH, in the postnatal setting, is now well established and offered as the first line test in most centres. Postnatal experience has also shown us, however, that with the additional diagnoses come less wanted findings, commonly known as Incidental Findings (IF) and Variants of Uncertain Significance (VOUS). In the UK, these issues have delayed the introduction of aCGH, in the prenatal setting, whilst the best way forward is considered. The importance of accuracy, speed and minimal diagnostic uncertainty is recognized, in the knowledge that such results may inform decisions about the outcome of a pregnancy. There is agreement that some tailoring of prenatal aCGH results is appropriate, and that such tailoring is best applied on a national basis but, as yet, no decision has been made on a certain way forward. This is currently under urgent review in the UK. The issues under discussion, and any conclusions drawn, will be presented.

Sagoo et al. 2009

Positive Array results after normal result on chromosome analysis

False Positive Array results after normal result on chromosome analysis

False Positives are similar to Positives Sagoo et al. 2009

>2300 children with CNV associated withintellectual disability and congenital anomalies

Girirajan et al NEJM September 2012

1

2 () (VOUS) ( )