Micro array analysis

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genetic prenatal diagnosis

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Micro array analysis

  1. 1. NARENDRA MALHOTRA M.D., F.I.C.O.G., F.I.C.M.C.H • Prof. Dubrovick International university,croatia • Indian FOGSI representative to FIGO • President FOGSI (2008) • Dean of I.C.M.U. (2008) • Director Ian Donald School of Ultrasound • National Tech. Advisor for FOGSI-G.O.I.—Mc Arthur Foundation EOC Course • Hon Prof Ob Gyn at DMIMS,Sawangi,Advisor ART unit at MAMC & SMS Jaipur • Editor od SAFOG journal • Chairman publication committee of AOFOG • Practicing Obstetrician Gynecologist at Agra. Special Interest in High Risk Obs., Ultrasound, Laparoscopy and Infertility, ART & Genetics • Member and Fellow of many Indian and international organisations • FOGSI Imaging Science Chairman (1996-2000) • Awarded best paper and best poster at FOGSI : 5 times, Ethicon fellowship, AOFOG young gyn. award, Corion award, Man of the year award, Best Citizens of India award • Over 30 published and 100 presented papers • Over 50 guest lectures given in India & Abroad.Presented 10 orations. • Organised many workshops, training programmes, travel seminars and conferences • Editor 8 books, many chapters, on editorial board of many journals • Editor of series of STEP by STEP books • Revising editor for Jeatcoate’s Textbook of Gynaecology (2007) and DONALD OBS MANNUAL(2012) • Very active Sports man, Rotarian and Social worker MALHOTRA NURSING & MATERNITY HOME 84, M.G. Road, Agra-282 010 Phone : (O) 0562-2260275/2260276/2260277, (R) 0562-2260279, (M) 98370-33335; Fax : 0562-2265194 E-mail : mnmhagra3@gmail.com RAINBOW HOSPITALS GLOBAL RAINBOW HEALTH CARE Consultant for IVF at jalandhar,ludhiana,ambala,bhiwani,gwalior,allahabad,gorakhpur,udaipur,bariely,jaipur,delhi Neapal & Bangladesh
  2. 2. Beyond ultrasound abnormalities what next ? and what more ?… time for FISH & CHIPS Narendra Malhotra,MD,FICOG FOGSI rep to FIGO Vaidehi Jobanputra, Ph.D., M.S., FACMG President , Advanced Genomics Institute & Laboratory Medicine (AGILE), New Delhi Jaideep Malhotra President elect ASPIRE Neharika Malhotra Bora Asst Prof BVP medical college,Pune
  3. 3. Nature, editorial Nov.2011
  4. 4. Prenatal Diagnosis for fetal anomalies • 20% of stillborn babies have a major malformation • 3% of live births have congenital anomalies • Diagnosis of a malformation syndrome requires • a number of “broad spectrum” analyses • followed by more sophisticated and targeted tests • Detailed ultrasound and invasive fetal testing • Etiology • Prognosis • Recurrence risk • Prevention / options in future pregnancies
  5. 5. Indications for Prenatal Diagnosis • Advanced maternal age • Previous child with de novo chromosomal aneuploidy – Woman 30 yrs, child with T21: Increased Recurrence risk for any chromosomal abnormality (1/100) versus age- related risk (1/390) • Parental structural chromosome abnormality • Family history of a genetic disorder • Elevated risk based on maternal serum screening • Fetuses with ultrasound examination
  6. 6. Its all in your “genes” and the genes are on your chromosomes
  7. 7. • Cytogenetics, the beginnings… • • 1956 Tjio and Levan • – human species has 46 chromosomes • • 1959 Léjeune • – Detection of the first chromosomal anomaly, trisomy 21 • • „Etude des chromosomes somatiques de neuf enfants mongoliens.“ • • 1963 Léjeune • – Diagnosis of first structural aberration, crit du chat syndrome, 5p- • • 1966 Steele and Berg • – fetal karyotyping on amniotic fluid • • 1968 Nadler et al. • – diagnosis of trisomy 21 in amniotic fluid • • 1984 Ward • – CVS in first trimester
  8. 8. Gold standard in prenatal analysis invasive procedures and conventional karyotyping
  9. 9. Chromosome Analysis Most widely used whole genome analysis method for clinical cytogenetics
  10. 10. Chromosomal analysis classical cytogenetics molecular cytogenetics 1960 1980 1990 2000 FISH Array- CGH Chromosome banding
  11. 11. Advances in invasive prenatal diagnosis: • 1.Chromosomal microarray (array-CGH, molecular karyotyping) versus conventional karyotyping to detect chromosomal aneuploidies • 2. Non-invasive analysis in maternal plasma • 3. Beyond chromosomal aberrations, diagnosis of monogenic disorders in prenatal analysis
  12. 12. Karyotyping versus microarray analysis Detects – large aberrations only (10Mb) – balanced translocations – triploidy • Does not detect – smaller aberrations Detects – large and small aberration, variants of unknown significance – unbalanced translocations • Does not detect – balanced translocations, triploidy
  13. 13. Chromosomal Microarray Analysis (CMA) AKA aCGH, SNP array, genomic microarray Compares differences in DNA copy number (dosage) between two genomes Single step genome scan Much higher resolution than chromosome analysis
  14. 14. A microarray is a multiplex lab-on-a-chip. It is a 2D array on a solid substrate (usually a glass slide or silicon thin-film cell) that assays large amounts of biological material using high- throughput screening methods. The concept and methodology of microarrays was first introduced and illustrated in antibody microarrays (also referred to as antibody matrix) in 1983 in a scientific publication and a series of patents.As the "gene chip" industry started to grow in the 1990's, with the establishment of companies, such as Affymetrix, Illumina, and others, the technology of DNA microarrays has become the most sophisticated and the most widely used
  15. 15. Types of microarrays include: DNA microarrays, such as cDNA microarrays, oligonucleotide microarrays and SNP microarrays MMChips, for surveillance of microRNA populations Protein microarrays Peptide microarrays, for detailed analyses or optimization of protein-protein interactions Tissue microarrays Cellular microarrays (also called transfection microarrays) Chemical compound microarrays Antibody microarrays Carbohydrate arrays (glycoarrays) Phenotype microarrays interferometric reflectance imaging sensor (IRIS)
  16. 16. A DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure theexpression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Each DNA spot contains picomoles (10−12 moles) of a specific DNA sequence, known as probes (or reporters or oligos). These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA (also called anti-sense RNA) sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target.
  17. 17. Four Types of Testing Programs • Newborn screening • Carrier testing • Prenatal testing • Presymptomatic (predictive) testing
  18. 18. Its now a story of FISH & CHIPS
  19. 19. Copy Number Changes • AKA Copy Number Variants (CNV) • Common cause of abnormal phenotypes – Congenital anomalies – Autism – Syndromic and nonsyndromic intellectual disability – Speech delay – Sezuires – Growth retardation with or without anomalies • Copy number changes are identified in ~20% of cases American College of Medical Genetics: CMA as first-line diagnostic test for postnatal cases
  20. 20. Prevalence of genomic changes in Fetuses with Selected Isolated and Multiple Sonographically Detected Abnormalities Abnormality Abnormal CMA (%) Isolated Multiple findings Cystic hygroma 52 71 Nuchal edema 19 45 Heart Defects 16 66 Duodenal atresia 38 64 Ventriculomegaly 2 17 Choroid Plexus cysts 1 48 Diaphragmatic hernia 2 49 Examphalos 8 46 Renal abnormalities 3 24
  21. 21. Amniotic fluid specimen 28 yrs old with absent nasal bone, increased NT Normal FISH for aneuplodies and Karyotype Case 1
  22. 22. Amnio 28 yrs old with absent nasal bone, increased NT Normal FISH and Karyotype
  23. 23. 52 Protein Coding Genes 14 Disease associated genes Common clinical features included feeding problems, behavioral problems, abnormally shaped teeth, scoliosis, developmental delay, MR. Deletion of chromosome 3 – 4.9Mb
  24. 24. CVS 31yrs old, fetus with Holoprocencephaly at 13 wks Case 2
  25. 25. 13 Disease associated genes including TRPS1 and EXT1, Langer-Giedion syndrome Common clinical features included in Langer-Giedion syndrome include multiple dysmorphic facial features, multiple cartilaginous exostoses, craniofacial and skeletal abnormalities Deletion on chromosome 8 – 20.9MbCVS 31yrs old with fetus with Holoprocencephaly
  26. 26. Amniotic Fluid Sample 25 yrs old with Abnormal Quad Screen Case 3
  27. 27. 35 Protein Coding Genes 6 Disease associated genes Common clinical features included ASD, VSD, developmental delay, MR, micrognatia, deafness Amniotic Fluid Sample 25 yrs old with Abnormal Quad Screen Duplication on chromosome 9: 9.1 Mb
  28. 28. 45 Protein Coding Genes 15 Disease associated genes including TCF4 Common clinical features included hypotonia, microcephaly, short strature, speech delay, cardiomyopathy, developmental delay, MR. Amniotic Fluid Sample 25 yrs old with Abnormal Quad Screen Deletion on chromosome 18 – 26.2Mb
  29. 29. • Patient elected to terminate the pregnancy • CMA suggests possible balanced translocation in the parents • Follow up parental karyotypes: – Mom 46,XX – Dad 46,XY,t(9;18)(p24;q21.2) • Increased risk for miscarriages and offspring with congenital abnormalities and/or MR Counseling
  30. 30. 40 yrs, AMA Case 4
  31. 31. 4 genes 1 Disease associated gene: STS, Ichthyosis X-linked Ichthyosis X-linked is a keratinization disorder manifesting with mild erythroderma and generalized exfoliation of the skin within a few weeks after birth Affected boys later develop large, polygonal, dark brown scales, especially on the neck, extremities and trunk Deletion on chromosome X– 1.15 MbAmnio 40 yrs, AMA
  32. 32. Challenges in CMA • Detection of balanced chromosomal rearrangements • Interpretation of CNV in the prenatal setting is more challenging than in the postnatal population • Whole Genome Scan provides data that requires careful clinical interpretation – Bioinformatic information vs Clinical correlations • Accurate counseling required when results of unknown clinical significance are discovered
  33. 33. Current Recommendations • American College of Obstetricians and Gynecologists (ACOG) and European Best Practice Guidelines do not yet include offering CMA to all patients undergoing invasive prenatal testing • ACOG has endorsed CMA an appropriate test in prenatal cases with abnormal ultrasound findings and a normal karyotype
  34. 34. Indications for CMA in Prenatal Diagnosis • Fetal structural anomaly: cardiac, central nervous system, skeletal, urogenital, renal, increased nuchal translucency and intrauterine growth restriction (IUGR) • Delineating marker chromosomes: origin and genomic content • Precisely determine the breakpoints of genomic alterations detected with conventional cytogenetic methods • Apparently balanced reciprocal balanced translocations
  35. 35. Indications for CMA in Still Births • About 5% of structurally normal stillborn fetuses have an abnormal karyotype • 35–40% of stillbirths that are structurally abnormal or macerated  Underestimates: poor quality of the karyotype  Tissue culture failure in 50% of cases
  36. 36. Summary • CMA is a new technology which provides a whole genome scan to detect copy number changes (CNCs) • Defines genomic aberrations with extraordinary precision and at “ultra-high” resolution • Prenatal CMA can detect clinically significant genomic aberrations in fetuses with U/S abnormality • Provide accurate diagnosis and prognosis both prenatally and postnatally to parents
  37. 37. summary • Advances in invasive prenatal diagnosis: Chromosomal • microarray (array-CGH, molecular karyotyping) will replace • conventional karyotyping • 2. Non-invasive prenatal analysis in maternal plasma will • replace other screening tests and further decrease the need • for invasive testing • 3. Diagnosis of monogenic disorders in prenatal analysis will • become feasible by panel testing • 4. Importance of pre- and post-test counseling, discussion • of ethical issues
  38. 38. Ethical dilemmas • • Prenatal diagnosis and abortion are major issues of moral sensitivity • • Molecular techniques allow narrowing or broadening of the scope of diagnostic testing • • Range of conditions tested and why? • • Testing marked driven / within public health care • • Basic principles of biomedical ethics • – respect for person, beneficience, non-maleficience, justice • – proportionality, equity of access, informed consent • • Ensure abortion decision remains personal, not turned into instruments of social goals
  39. 39. Acknowledgements our partners in GENETIC ANALYSIS & DIAGNOSIS Contact Info:: Email: vaidehi@labassure.com Website: www.labassure.com info@rainbowhospitals.org rainbowgenetics@rainbowhospitals.org yourgenes@rainbowhospitals.org

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