DrDianatpour Medical Geneticist Prenatal tests Invasive Karyotype FISH QF PCR Array CGH Noninvasive NIPT Karyotyping Chromosome Preparation Any tissue with living nucleated cells that undergo division can be used for studying human chromosomes Most commonly circulating lymphocytes fro ID: 934787
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IN THE NAME OF GOD
Genetic Tests for Detection of Chromosome Abnormalities in Perinatology
Dr.Dianatpour
Medical Geneticist
Slide2Prenatal tests
InvasiveKaryotypeFISHQF PCRArray CGHNoninvasiveNIPT
Slide3Karyotyping
Chromosome Preparation Any tissue with living nucleated cells that undergo division can be used for studying human chromosomes. Most commonly circulating lymphocytes from peripheral blood are used, although samples for chromosomal analysis can be prepared relatively easily using skin, bone marrow, chorionic villi, or cells from amniotic fluid (amniocytes
).
Slide4Slide5Rate of Chromosome abnormalities in amniotic fluid karyotype (cut off 1:250)Trisomy (21,18,13) 3.4%
All aneuploidies 3.9%Translocation 0.5Normal variation 6.3
Slide6Normal variationInv(9)(p12q13) Pericentric
inversion of chromosome 9 9qh+ Increase in the length of heterochromatin on the long arm of chromosome 9Other hetereomorphysms( 1qh+, 16qh+ and Yqh+)
Slide7Normal variation
15ps+ Increase in the length of the satellite on the short arm of chromosome 15
Slide8Heteromorphisms clinical significanceThe
study by Cheng et al., (2017) is the largest published study to date (n = 19,950) Most studies have reported that the incidence of heteromorphisms is three- to five-fold higher in the infertile population compared with fertile individuals. Current prevailing hypotheses of how these variants could impact fertility include altered regulation of the genome and aberrations in chromosome pairing and cell division.
Can this information be utilized clinically?
Slide9Parents with Balanced translocationDiagnostic tests should be requested, Karyotype or Array CGH.
NIPT Should not be requested
Slide10Previous child with down syndromeKaryotypeNIPT
Slide11Structural abnormalityCan chromosome Structural abnormality be screened by Maternal Serum screening tests ??
Slide12Molecular karyotypingFISH (Fluorescent insitu
Hybridization) Array CGH (Comparative Genome Hybridization)QF-PCR (Quantitative Fluorescent PCR)MLPA (Multiplex Ligation Probe Amplification)
Slide13Slide14FISHAdvantagesDisadvantages
Slide15QF PCRUninformative markersLow level mosaicismQF karyotype discrepancy
Slide16Chromosomal Microarray Analysis (CMA)CMA is a method of
measuring gains and losses of DNA throughout the human genome. It can identify chromosomal aneuploidy and other large changes in the structure of chromosomes that would otherwise be identified by standard karyotype analysis, as well as submicroscopic abnormalities
that are too small to be detected by traditional modalities
Slide17Chromosomal Microarray Analysis (CMA)In the field of prenatal diagnosis, the probability of finding a pathogenic copy number variant
is highly correlated with the presence of structural fetal abnormalities, although significant copy number variants also can be identified in structurally normal fetuses.
Slide18Copy number variantsUnlike aneuploidies, copy number variants are independent of maternal age and occur in approximately 0.4% of pregnancies
. Therefore, based on a systematic review, pregnancies in patients under 36 years of age have a higher risk for microarray abnormalities than for trisomy 21.Patients who prefer comprehensive prenatal detection of as many chromosomal aberrations as possible should be offered diagnostic testing and CMA. BAC array vs Oligo array
Slide19Limitations of CMA Balanced
chromosome rearrangements (eg, inversions or translocations), which do not result in deletion or duplication of genetic material. low-level mosaicism.variants of uncertain clinical significance.
Array CGH can not detect triploid fetus.
Slide20CMA: pretest and post test counsellingLate onset diseases like CMT
variants of uncertain clinical significance.
Slide21CMA vs Karyotypingchromosomal microarray analysis identified additional clinically significant abnormalities
in approximately 6% of fetuses with ultrasonographic abnormalities and a normal conventional karyotype result. Further, microarray analysis detected an abnormality in 1.7-2.0% of fetuses with an abnormal screening test result and a normal karyotype result
Slide22variants of uncertain significance (VUS)
Prenatal tests of all types, including ultrasonography, screening tests, and diagnostic tests, can provide results of uncertain significance. When so-called genetic “variants of uncertain significance” (VUS). Referral to a genetics expert for consultation and counseling can assist the patient with informed decision making.
Slide23Cell-Free DNA Screening
Cell-free DNA screens for aneuploidies using the analysis of cell-free DNA fragments in the maternal circulation starting at about 9–10 weeks of pregnancy and, unlike analyte screening, can be sent until term. The fetal component of cell-free DNA is derived from placental trophoblasts that are released into the maternal circulation from cells undergoing programmed cell death. The fetal component is known as the fetal fraction; it comprises approximately 3–30% of the total cell-free DNA in maternal blood (Average : 10-15%)
Slide24Fetal FractionThe quantity of cell- free DNA from the fetal component increases throughout gestation. The quantity of the fetal fraction is affected by many factors, including but not limited to
gestational age, maternal body mass index (BMI), maternal medication exposure, maternal race,
aneuploidy status if present, fetal or maternal mosaicism, and singleton or multiple gestation (13–18). Depending on the laboratory, cell-free DNA screening can be performed as early as 9 weeks of gestation, although higher fetal fractions at 10 weeks and beyond are associated with lower rates of test failure.
Slide25Cell-free DNACell-free DNA is the most sensitive and specific screening test for the common fetal aneuploidies.
Nevertheless, it has the potential for false-positive and false-negative results. Furthermore, cell-free DNA testing is not equivalent to diagnostic testing. The sex chromosome results for patients who have undergone organ transplantation
will be affected by the sex of the organ donor and therefore sex chromosome testing is not recommended in this population.
Slide26Detection rateThe most recent meta-analysis evaluating test performance for cell-free DNA screening (19) reports a greater than
99% detection rate for fetal trisomy 21, 98% detection rate for fetal trisomy 18, and fetal trisomy 13 with a false-positive rate of 0.13%; Patients whose cell-free DNA screening test results are not reported by the laboratory or are uninterpretable (a no-call test result) are at increased risk for chromosomal abnormalities.
Slide27Other Potential Chromosomal Abnormalities Identified by Cell-Free DNA In
addition to screening for the common aneuploidies, some laboratories offer testing for other aneuploidies such as trisomy 16 and trisomy 22, microdeletion testing, and genome-wide screening of large copy number changes (26–28). Nonmosaic fetal trisomy 16 or 22 is associated with a nonviable gestation. Mosaic trisomy 16 and 22 can be associated with fetal survival; however, screening is not recommended because the screening accuracy with regard to detection and the false-positive rate is not established.
Slide28How does screening for chromosomal abnormalities differ in twin gestations?
No method of aneuploidy screening that includes a serum sample is as accurate in twin gestations as it is in singleton pregnancies In a dizygotic twin pregnancy, a screen positive test infers that at least one of two fetuses would be aneuploid. This assumes that monozygotic pregnancies have equivalent trisomy 21 risk per pregnancy relative to maternal age-matched singletons and dizygotic pregnancies have twice the risk of at least one affected fetus
.
Slide29Twin gestationsA recent meta-analysis suggests that
first‐trimester combined screening in twins has a detection rate of 89% with a false-positive rate of 5.4%, which is similar to singleton gestations (64). Although serum screening evaluates the pregnancy as a whole, the NT measurement directly evaluates the individual fetus.
Slide30Cell-free DNA in Twin gestations
Twin fetuses in a single pregnancy each contribute different amounts of cell-free DNA into the maternal circulation. It is possible that an aneuploid fetus would contribute less fetal DNA, therefore masking the aneuploid test result. Recent studies have suggested that sensitivity for trisomy 21 with cell-free DNA in twin pregnancies may be similar to singletons when a test result is returned; however, there is a higher rate of test failure
(45, 67).
Slide31Atypical chromosomal abnormalitiesAtypical chromosomal abnormalities were defined as major abnormalities not detectable on standard five‐chromosome NIPT, that is, excluding T21, T18, T13, monosomy X and sex chromosome
trisomies.
Slide32Atypical chromosomal abnormalitiesOne population‐based study has reported an increased risk of atypical chromosome abnormalities in women with low maternal serum
markers (PAPP‐A) or (β‐hCG) < 0.2 MoM, independent of cFTS risk result.
Slide33The potential impact of NIPT
ResultsAbnormal karyotype results were reported in 224 of 1488 (15%) women with high‐risk pregnancies having invasive diagnostic testing. NIPT potentially would have identified 85%. The 33 abnormalities unidentifiable by NIPT were triploidies (n = 7, 21%),
balanced (n = 8, 24%) and unbalanced rearrangements (n = 10, 30%) and level III mosaicisms (n = 8, 24%).
Slide34Take home message11- prenatal chromosomal microarray analysis is recommended for a patient with a fetus with one or more major structural abnormalities identified on
ultrasonographic examination and who is undergoing invasive prenatal diagnosis.
Slide35Take home message22-One population‐based study has reported an increased risk of atypical chromosome abnormalities in women with low maternal serum markers (PAPP‐A) or (β‐
hCG) < 0.2 MoM, independent of cFTS risk result.MOM in twin pregnancy?
Slide36Take home message33-R
eplacing cFTS with NIPT for T21 screening will lead to a decline in the 11–13‐week nuchal translucency (NT) ultrasound and resultant missed opportunities for early detection of fetal structural malformations.
Slide37Take home message44-There are additional concerns that the use of NIPT as a secondary screening test, i.e. the so‐called ‘contingent’
model may lead to a reduction in the detection of atypical chromosome abnormalities identified previously through diagnostic testing after a result of high risk on CFTS.
Slide38Take home message5Contingent vs stepwise maternal serum screening
Contingent
Slide39Thank you