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Director Italian Branch Cagliari Regional Director for Europe

WHO Collaborating Centre for Community Control of Hereditary Diseases. Director Italian Branch Cagliari Regional Director for Europe. Director Ian Donald School for Invasive Procedures. INVASIVE VS NON-INVASIVE PRENATAL DIAGNOSTIC PROCEDURES Giovanni Monni.

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Director Italian Branch Cagliari Regional Director for Europe

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  1. WHO Collaborating Centre for Community Control of Hereditary Diseases Director Italian Branch Cagliari Regional Director for Europe Director Ian Donald School for Invasive Procedures INVASIVE VS NON-INVASIVE PRENATAL DIAGNOSTIC PROCEDURES Giovanni Monni 12th TURKISH GYNECOLOGY AND OBSTETRICS CONGRESS Antalya, 15th – 19th Maggio 2014

  2. DILEMMAS TO AVOID GENETIC DISORDERS IN THE NEWBORNS • Screening based on maternal age alone? • First or second trimester ultrasound and biochemical screening? • Prenatal invasive procedures? • Standard karyotype? aCGH analysis? • Preimplantation genetic diagnosis? • Diagnostic ultrasonography (1st-2nd trimester)? • Fetal cell free-fetal DNA (cff- DNA) in maternal blood (general or contingent) ?

  3. CHANGES IN THE APPROACH FOR INVASIVE PRENATAL DIAGNOSIS IN 35,127 CASES AT A SINGLE CENTER FROM 1977 TO 2004 • DIAGNOSI SEMPRE Più PRECOCE Monni, Fetal Diagn Ther 2006

  4. Number of amniocenteses and chorionic villus samplings carried out in Denmark, 2000-2006 Ekelund, BMJ 2008

  5. Total Number of Diagnostic Procedures in England (2003- 2012) Morgan,UOG 2013

  6. UK national policy study ofaneuploidy screening after the implementation of the combined test • Reduction of false positive rate from 6% to 3% without significant change of DR of Down Syndrome • Progressive reduction in the number of screen-positive cases • Significant reduction in the number of invasive prenatal diagnostic procedures Morgan, Ultrsound Obstet Gynecol 2013

  7. UK NATIONAL POLICY STUDYANEUPLOIDY SCREENING The odds of the fetus being affected after a positive combined test in the first trimester were much greater than were the odds based on advanced maternal age alone (1:20 vs 1:75).So a significantly higher probability of an invasive test would confirm an abnormal fetal karyotype. Morgan, Ultrsound Obstet Gynecol 2013

  8. REDUCTION IN THE FETAL NUMBER OF INVASIVE PROCEDURES PERFORMED FOR PRENATAL KARYOTYPE Redistribution of the proportion of procedures performed by amnio and CVS- Denmark: in 2006 CVS in 66% of cases- UK: in 2003 Amnio/CVS 3:1 in 2011 Amnio/CVS 1:1 Monni, Zoppi, Ultrasound Obstet Gynecol 2013: Opinion

  9. FIRST TRIMESTER EUROPEAN NATIONAL POLICY FOR PRENATAL DOWN SYNDROME SCREENINGDenmark (BMJ 2008) and UK (UOG 2013) Studies - Decrease in Fetal Loss due to a reduction in invasive diagnostic procedures- Earlier Diagnosis of Chromosomal Aneuploidies Monni, Zoppi, Ultrasound Obstet Gynecol 2013: Opinion

  10. Nicolaides, Prenat Diagn 2012 FIRST TRIMESTER SCREENING AND INVERSION OF THE PYRAMID OF PRENATAL CARE

  11. INVASIVE PRENATAL DIAGNOSIS TECHNIQUE OF 78 CHROMOSOMAL ABNORMALITIESOSPEDALE MICROCITEMICO- CAGLIARIJANUARY 2011 – DECEMBER 2011

  12. Distribution of number of fellows for CVS training at the Ospedale Microcitemico - Cagliari * BEFORE NT SCREENING ** AFTER NT SCREENING

  13. FELLOWS TUTORED AT MICROCITEMICO HOSPITAL IN CAGLIARI (No 151) Other: Argentina, Azerbaijan, Bosnia, Czech Republic, Canada, Japan, France, Germany, India, Lebanon, Mongolia, Morocco, Netherlands, Portugal, Romania, S. Arabia, Slovenia, Spain, Sudan, Un. Arab Emirates, Venezuela

  14. NEW LABORATORY TECHNIQUES • Fluorescent in situ hybridization (FISH) • Amplification of polymorphic chromosome-specific markers by polymerase chain reaction (PCR) • Most laboratories offer a rapid test (PCR or FISH) to detect trisomy 21, 18, 13 and sex chromosome aneuploidies, as well as tissue culture to provide a full karyotype • Array comparative genomic hybridization (a-CGH): in cases of multiple congenital abnormalities at ultrasound or for clinical diagnosis?

  15. Advantages of array Comparative Genomic Hybridization (aCGH) or Chromosomal Microarray Analysis (CMA) • aCGH allows detection of smaller pathogenic chromosomal variants that are undetectable using standard cytogenetic analyses (G-band karyotyping)

  16. DISADVANTAGES OF ACGH • aCGH does not allow detection of balanced chromosomal rearrangements triploidy and some instances of mosaicism • The biggest challenge presented by aCGH is the detection of chromosomal variants of unknown clinical significance (VOUS)

  17. METHODS FOR ANALYSIS OF CELL-FREE (CF) DNA IN MATERNAL BLOOD • Shotgun massively parallel sequencing (s-MPS) • Targeted massively parallel sequencing (t- MPS) • Single nucleotide polymorphism (SNP) -based analysis

  18. CFDNA ANALYSIS FOR T21: A META-ANALYSIS (18 CITATIONS 2011- 2013) • Individual studies: • Detection Rate (DR) ranges: 94.4-100% • False Positive Rate (FPR) ranges: 0- 2.05% • Pooled weighted: • DR: 99.0% (95% CI 98.2- 99.6) • FPR: 0.08% (95% CI 0.03- 0.14) Gil et Nicolaides, Fetal Diag Ther 2014

  19. CFDNA ANALYSIS FOR T21: A META-ANALYSIS Gil et Nicolaides, Fetal Diag Ther 2014

  20. CFDNA ANALYSIS FOR T18, 13, MONOSOMY X : A META-ANALYSIS The poor performance of cfDNA analysis in screening for trisomy 13 and monosomy X could be due to the highly variable amplification of chromosome X and 13 because of a lower guanosine- cytosine content Gil et Nicolaides, Fetal Diag Ther 2014

  21. CFDNA ANALYSIS FOR SEX CHROMOSOME ANEUPLOIDIES OTHER THAN MONOSOMY X • Pooled weighted: • DR: 93.8% (95% CI 85.9- 98.7) • FPR: 0.12% (95% CI 0.02- 0.28) Gil et Nicolaides, Fetal Diag Ther 2014

  22. CFDNA ANALYSIS FOR TRIPLOIDY • Diandric (paternal): • Placenta enlarged and partially molar • NT enlarged • Free- beta hCG very high (10 times higher than normal) • Digynic (maternal): • Placenta very small • Fetus severely growth restricted • Normal NT • Free- beta hCG and PAPP-A very low The SNP method for cfDNA testing is the only one at present that can detect triploidy because it analyses allele distributions 4 out 8 cases of diandric triploidy have been detected, and suspicion raised for a case of diagynic triploidy Utility of cfDNA as first-line method of screening because identification of triploidy would be beneficial (diandric triploidy can cause maternal complications including early- onset preeclamsia and choriocarcinoma) Gil et Nicolaides, Fetal Diag Ther 2014

  23. LIMITATIONS OF CFDNA TESTING • Failure to provide results • Receiving results in 1- 2 weeks • Cost

  24. FAILURE TO PROVIDE RESULTS In 1- 5% of cases no results is given after first sampling • Problems with sample collection or with transportation to the laboratory (on repeat sampling result is obtained in about 100%) • Assay failure (on repeat sampling result is obtained in about 75%) • Low fetal fraction (on repeat sampling result is obtained in about 50%); if it is a consequence of maternal obesity this problem is difficult to overcome Gil et Nicolaides, Fetal Diag Ther 2014

  25. RECEIVING RESULTS IN 1- 2 WEEKS • Average interval 10 calendar days • In 95- 98% of cases a result available within 14 days • In 2% of cases a result may not be available in less than 3-4 weeks Such delay may reverse the beneficial shift in screening and diagnosis of aneuploidies from the second to the first trimester Gil et Nicolaides, Fetal Diag Ther 2014

  26. MODELS FOR CLINICAL IMPLEMENTATION OF CFDNA TESTING IN MATERNAL BLOOD • Routine screening for whole population • Contingent screening based on the result of first trimester combined test

  27. MODELS FOR CLINICAL IMPLEMENTATION OF CFDNA TESTING AS FIRST-LINE METHOD FOR ALL PREGNANCIES • 10 weeks, maternal blood to all • 12 weeks first trimester us Expected: • 99% DR of trisomy 21 • 95% DR of trisomy 18 and 13 • 1% Invasive testing rate Gil et Nicolaides, Fetal Diag Ther 2014

  28. MODELS FOR CLINICAL IMPLEMENTATION OF CFDNA TESTING AS CONTINGENT SCREENING HIGH RISK GROUP • Maternal blood in the high risk group (> 1:100) Expected: • 86% DR of tris. 21 • 89% DR of tris.18 /13 • 0.4% Invasive test. rate cfDNA testing could not detect other aneuploidies Gil et Nicolaides, Fetal Diag Ther 2014

  29. MODELS FOR CLINICAL IMPLEMENTATION OF CFDNA TESTING AS CONTINGENT SCREENING INTERMEDIATE RISK GROUP • Maternal blood in the Intermediate Risk Group (>1:11<1:2,500) Expected: • 97.6% DR of tris. 21 • 98.1% DR of tris. 18/13 • 0.8% Invasive test. rate cfDNA testing could not detect other aneuploidies Gil et Nicolaides, Fetal Diag Ther 2014

  30. PRENATAL NONINVASIVE DIAGNOSIS FOR MONOGENIC DISEASE: ACTUALLY VALIDATED USE • Fetal sex determination (X-linked diseases in order to avoid invasive procedure in female fetuses) or for congenital adrenal hyperplasia (CAH) for therapeutic options • RH blood group, D antigen • Paternal inherited autosomal dominant diseases or de- novo after ultrasound suspicion (chondrodysplasias) SIGU 2014, Document on the indications of use of performing non-invasive prenatal research

  31. PRENATAL NONINVASIVE DIAGNOSIS FOR MONOGENIC DISEASE: NOT YET VALIDATED USE • Autosomal recessive diseases • X linked diseases • Autosomal dominat diseases of maternal origin SIGU 2014, Document on the indications of use of performing non-invasive prenatal research

  32. MAIN FEATURES OF FREE DNA IN MATERNAL PLASMA • Free DNA is always present in peripheral blood with a magnitude of between 145 and 201 bp • Pregnancy causes an increase in the size of circulating DNA of maternal origin and a progressive increase in the concentration of Fetal DNA that is smaller • The origin of circulating Fetal DNA in maternal plasma is due to placental apoptotic processes of the syncytium trophoblast • The Fetal DNA is present since the 7th week of pregnancy and increases during pregnancy. In 10 weeks increases to about 5 or 10% of the total circulating plasma DNA. The fraction of fetal tissue correlates negatively with the maternal weight • The presence of Fetal DNA in maternal plasma is no longer detected two hours after giving birth. The average half-life of 16.3 minutes (range 4-30 minutes)

  33. FEASIBILITY STUDY OF -THALASSEMIA NIPD IN SARDINIA BY BENCHTOP NEXT GEN SEQUENCING APPARATUS (PGM LIFE TECHNOLOGIES)

  34. LIBRARY PREPARATION (51 AMPLICONS) Chr 11 Cluster HBB = 48 amplicons (85-197 bp)  G    A HINC II HIND III HINC II Ava II BamHI TSPY (175 bp) ZFX SRY Chr Y ChrX (88 bp) (139 bp) ZFY (88 bp)

  35. TAKE HOME MESSAGES (1) • Maternal age should no longer be the sole criterium for set the parental choice of invasive prenatal diagnosis • First trimester combined screening reduces the number of invasive prenatal diagnostic procedures • After a positive combined test, a significantly high probability of an invasive test would confirm an abnormal fetal karyotype • First trimester combined test induces reversing the traditional pyramid of prenatal care • Educational organizations have faced new challenges in providing training for invasive procedures

  36. TAKE HOME MESSAGES (2) • aCGHisnot a substituteforconventionalkaryotyping; 2) aCGHshouldbeusedforspecificdiagnosticpurposes in selectedpregnancies and notforgeneral screening in allpregnancies;

  37. TAKE HOME MESSAGES (3) 1) cff- DNA for NIPT has the role of a screening test 2) Evidence from high risk population 3) Necessity of implementation of cff- DNA in low risk series 4) Genetic counselling is mandatory before and after NIPT

  38. NON-INVASIVE PRENATAL TEST (NIPT) • The expectations regarding cff-DNA for fetal genetic anomalies are very high because it may have the potential to change the landscape of prenatal diagnosis. However, to the disappointment of many, cff-DNA does not have the ability to function as a diagnostic test but is considered at present time as a • “super” screening test. Monni, Journal of Perinatal Medicine 2014

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