Applications of Chromosomal Microarray Analysis (CMA) in pre- and postnatal Diagnostic: advantages, limitations and concerns جواد کریمزاد حق PhD of Medical Genetics آزمايشگاه پاتوبيولوژي و ژنتيك پارسه
Array CGH (comparative genomic hybridization)/ (single nucleotide polymorphism) SNP Array I:Technical Principles II: Clinical Applications
I:Technical Principles Array-CGH, SNP-Array
acgh: Array comparative genomic hybridization (also microarray-based comparative genomic hybridization, array CGH, acgh) is a molecular cytogenetic technique for the detection of chromosomal copy number changes on a genome wide and high-resolution scale.
SNP array is a type of DNA microarray which is used to detect polymorphisms within a population. A single nucleotide polymorphism (SNP), a variation at a single site in DNA, is the most frequent type of variation in the genome.
Differences in the ability to resolve genomic changes between these arrays I:Array CGH Gain Detection of CNVs Loss Detection of CNVs (Unbalanced chromosomal rearrangements) single-exon copy number variants (CNVs) Resolution 8x60k, 4x180k, 2x400k, 1M SurePrint G3 Custom CGH+SNP (LOH-Analayse, Delectation of UPD) Agilent II: SNP Array Gain Detection of CNVs Loss Detection of CNVs (Unbalanced chromosomal rearrangements) Determining disease susceptibility (Genome wide association study) Loss Of Heterozygosity (LOH) higher sensitivity for the detection of low-level mosaic aneuploidies and chimerism indicate uniparental disomy (UPD) Detection of Polyploidy, MaternalContamination Parental DNA needed!! Resolution 2.7 M, 1.8M (SNP 6.0) Affymetrix
DNA Extraction To Annotation DGV/DECIPHER/ISCA/UCSC/OMIM, Gene Reviews and recently published reviews Benign (Literature reports! no genes!! enriched in repetitive sequences, short size), Definitely pathologic (Literature reports! Dom. Pattern (HI), high densities of functional elements, large size) Uncertain clinical Significance (likely pathologic: small database reports, one gene!//likely benign: small databases polymorph in general population// unclassified: Dosage effect of, genes is unknown, Inherited Incidental Finding (for. example: risk of neoplasia :TP53,RB) Difficult Interpretation by high resolution(by high resolution)
Copy Number Variations (CNVs) 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 ~ 4.8-9.5% of the human genome can be classified as copy number variations and approximately 100 genes that can be completely deleted without producing apparent phenotypic consequences. Nature Reviews Genetics. 16: 172 183
II: Clinical Applications
The majority (>95%) of benign CNVs in humans are <100 kb in size
Clinical Applications 1. PGD/PGS/PND 2. Abortion 3. Ultrasonographic detected structural abnormalities of fetus 4. non-syndromic developmental delay/intellectual disability (DD/ID), Multiple Congenital Anomalies (MCA) not specific to a well-delineated genetic syndrome, Autism Spectrum Disorders (ASD) and children with growth retardation and speech delay
1. PGD/PGS/PND PGS identifies chromosomally normal embryos ("euploid" embryos) for transfer to the uterus in the last step of an IVF cycle. Patients with a history of recurrent miscarriage or failed IVF cycles, which can be linked to chromosomal abnormality, may benefit from PGS. In addition, patients may also undergo PGS with the goal of transferring a single embryo. Blastomer biopsy of a 3-day-old embryo Trophectoderm biopsy of a 5-day-old embryo
2. Abortions In about 50% of first trimester spontaneous abortion the cause remains undetermined after standard cytogenetic investigation. array-cgh: detects abnormalities in a further ~10% of spontaneous abortion specimens considered to be normal using standard cytogenetic methods Spontaneous abortion and stillbirth by array-based comparative genomic hybridization 43 fetuses with spontaneous abortion and stillbirth. 32 (74.4%) of the samples, which included 26 (60%) aneuploidies and 10 (23%) pathogenic CNV. 2015 Jun;32(3):348-52. doi: 10.3760/cma.j.issn.1003-9406.2015.03.010.
3. Ultrasonographic detected structural abnormalities of fetus Chromosomal Microarray Analysis (CMA) can identify also imbalanced chromosomal aberration in approximately 6% of fetuses with ultrasonographic abnormalities with a normal karyotype result. Anencephaly Microcephaly Cleft palate Polyhydramnios Encephalocele Cystic hygroma Hydrops fetalis Dandy-Walker malformation Polydactyly Holoprosencephaly Oligohydramnios
Result: arr[hg19] 22q11.21(18628073_21857001)x3 Microarray CGH The gain is between locus q11.21 starting from 18628073bp to 21857001bp ranging 3.23Mb.
Two breakpoints between USP18 and CLTCL1 locus in the centromer section and LZTR1 and HIC2 locus in the telomeric section, that contains a genome duplication sizing about 2-3 Mb. This microduplication was also identified exactly in the father of the fetus.
4. non-syndromic developmental delay/intellectual disability (DD/ID), multiple congenital anomalies (MCA) not specific to a well-delineated genetic syndrome, autism spectrum disorders (ASD) and children with growth retardation and speech delay Array CGH is recommended as the first-tier evaluation in individuals with developmental disabilities or congenital anomalies not specific to a well-delineated mono genetic syndrome. The value of acgh screening as a first-tier test in children with multiple congenital anomalies has been studied and consensus adopted. Disease-causing CNVs are found in 5% 10% of cases of autism. Among 1792 patients (DD/MCA/ASD), 13.1% had clinically relevant results, either abnormal (n 131; 7.3%) or variants of possible significance (VPS; n 104; 5.8%). Genetics IN Medicine Volume 13, Number 9, September 2011
Confirm of array results /FISH /Realtime-PCR or PCR-capillary
Conclusion Array CGH/SNP Indications Example I: A parents with known balanced Translocation or marker-chromosome or a ring X-chromosome, Inversion wit an affected person, de novo translocation (8-10%) (one of parents has an chromosomal rearrangement without clinical features) PGD/PGS/CVS/Amniocentesis/cordocentesis Example II: IVF Failures, Recurrent pregnancy loss, Abortions PGS/POC Example III: Fetus with structural anomalies IUFD, IUGR : QF- PCR, FISH, MLPA and karyotype results are normal! Example IV: ID/DD, ASD, MCA Prenatal diagnostic Postnatal diagnostic For Choosing of suitable Array resolutions the physicians should be counseling with Genetics-Laboratories (Examples: 60 k for Abortion, 180 k for ASD)