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Genomic Approaches to Reproductive Disorders

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Genomic Approaches to Reproductive Disorders Genomic Approaches to Reproductive Disorders Aleksandar Rajkovic Dept Obstetrics Gynecology and Reproductive Sciences University of Pittsburgh Magee Womens Research Institute Pittsburgh, PA Preconceptional Care Scope • Half of Pregnancies are Unintended • Medical Conditions • Mental Conditions • Immunization History • Nutritional Issues • Family History/Genetic Risk • Occupational/Environmental Exposures • Tobacco/Drug Abuse • Social Issues Preconceptional genetic screening Ethnic: Sickle cell disease Tay–Sachs disease Pan-ethnic: cystic fibrosis fragile X syndrome Spinal muscular atrophy Mendelian Inheritance • 5593 phenotypes for which molecular basis known • 3452 genes with phenotype causing mutation • Over 15,000 mutations to date known Preconceptional Pan Ethnic Testing • Screens for known mutations in more than 100 genes, easy on genetic counsellors • The screen is pan-ethnic • Useful also for couples undergoing IVF and potentially PGD • 1:5 will be carriers of a Mendelian disorder. • $600 (529 Euros) for the couple Genetic Counselling • Objective of the test • Test Methodology • Type of sample required (parents, siblings) • Possible outcomes (abnormal results, result of unknown clinical significance) ClinVar Stars and their interpretation Number of golden stars No submitter provided an interpretation with assertion criteria (no assertion criteria provided), none or no interpretation was provided (no assertion provided) At least one submitter provided an interpretation with assertion criteria (criteria provided, single submitter) or multiple submitters provided one assertion criteria but there are conflicting interpretations in which case the independent values are enumerated for clinical significance (criteria provided, conflicting interpretations) Two or more submitters providing assertion two criteria provided the same interpretation (criteria provided, multiple submitters, no conflicts) three reviewed by expert panel four practice guideline De novo genomic events • Meiotic errors (Aneuploidies) • Mitotic errors (Mosaicisms) • De novo deletions or duplications (DiGeorge Syndrome) • De novo mutations (Tuberous sclerosis, Neurofibromatosis) Maternal age effects on incidence of trisomies Hassold and Hunt, Nat Rev Genet, 2001 Chaos in the Embryo Vanneste et al, Nature Medicine, 2009 PGS at UPMC Embryo biopsy Microarray PGS Array-CGH Precursor placenta cells DNA, extraction, amplification Embryo Precursor fetal cells Simultaneously tests for all 24 chromosomes Embryo 1: Male -Normal Male Embryo 2: Female - Trisomy 21 Female Embryo 3: Male – Monosomy 11 Male Embryo 4: Male Monosomy 15 Male Indications for PGS • Recurrent miscarriage • AMA • Diminished ovarian reserve • Multiple failed IVF • Personal reasons • Improve singleton pregnancy IVF • Reduction of Twins Post-implantation testing First Trimester Screening 1. Nuchal Translucency 2. PAPP-A 3. Free-ßHCG Detects Trisomy 21, 18 and 13 90% detection rate at 5% false positive rate Cell Free DNA based Screening 1893 Schmorl Trophoblasts in Maternal Pulmonary Vasculature 1948 Marked the identification of DNA in peripheral blood by Mendel and Métais 1959 Douglas Circulating Trophoblasts 1969 Walknowska XY Lymphocytes of Pregnant women with a Male Fetus 1979 Herzenberg Isolated Fetal Cells with FACS 1989 Lo Amplified Fetal DNA from Cells in Maternal Blood 1990 Bianchi Fetal Erythroblasts isolated from maternal blood using FACS 1992 Bianchi FISH for Aneuploidy of Fetal Erythroblasts 1992 CacheuxFISH for Aneuploidy of Fetal Trophoblasts 1994 Ganshirt Fetal Cells isolated using Magnetic-Activated Cell Sorting (MACS) 2008/2009 Quake, Lo and Peters publish independently on the massive parallel sequencing and its utility Facts about Cell Free Fetal DNA • Short DNA fragments, less than 200 bp • Represents a small fraction of cfDNA • Earliest day 18 after embryo transfer • Placenta is the origin of most of the fetal cfDNA (apoptosis, necrosis, remodeling) • Following delivery, cleared rapidly with half-life of 16 minutes Cell free prenatal DNA screening test General Principle of Non-Invasive Detection of Trisomy By Shotgun Sequencing Chromosome 1,2,3…..20,22 Chromosome 21 Normal 2:2 inter-chromosomal tag ratio Chromosome 1,2,3…..20,22 Chromosome 21 Trisomy 21 2:3 inter-chromosomal tag ratio Fetal aneuploidy is detectable by the overrepresentation of the affected chromosome in maternal blood Fan H C et al. PNAS 2008;105:16266-16271 ©2008 by National Academy of Sciences Importance of Fetal Fraction 1. Fetal DNA comprises up to 30% of circulating Cell-Free DNA 2. Fetal circulating Cell-Free DNA in 150-300 bp lengths (human genome is ~3.2 Billion base pairs long) 3. Between 10-20 weeks: a. Fetal Fraction is generally 10-15% b. 1-3% of samples will have fetal fraction of <4% c. In some cases repeat sampling will yield a higher fraction 4. Appears not to change after CVS or amnio Current Indication: High Risk •Maternal age 35 years or older at delivery •Suspicious fetal ultrasonographic findings •History of a prior pregnancy with a trisomy •Positive first trimester or second trimester screening •Balanced robertsonian translocation with increased risk of fetal trisomy 13 or trisomy 21. Clinical Trials (>7): Non-Invasive Prenatal Testing* Detection Rate False Positive Rate Trisomy 21 99.5% 0.2% Trisomy 18 98.4% 0.2% Trisomy 13 84.6% 0.9% * No result obtained in approximately 4% of women Can We Detect Subchromosomal Abnormalities Noninvasively? Genomic resolution 1 base pair (bp) to 10 megabase (Mb) G banding > 4 Mb FISH 40 to 250 kb /clone Chromosomal microarray 1kb DNA sequence [1 bp] Multiple congenital anomaly • A 25 year old gravida 5 para 3104 had anatomy scan at 19 weeks. • Ultrasound: Left sided diaphragmatic hernia with the heart pushed to the right side of the chest, thickened nuchal fold of 7 mm, echogenic kidneys bilaterally. • NORMAL KARYOTYPE A 1.4 Mb deletion on 17q12 MAA and Karyotype Results 177 samples Normal Unclear Incidental Cultural Abnormal significance findings artifacts 135 (76.3%) 16 (9%) 4 (2.3%) 2 (1.1%) 20 (11.3%) Abnormal Cultural Normal Abnormal Karyotype artifacts Karyotype karyotype 1 (0.6%) 1 (0.6%) 165 (93.2%) 10 (5.6%) Yatsenko et al, Clinical Genetics, 2013 Case • Couple with a child who had intellectual disability, short stature and dysmorphic features. • Child inherited a 4.2-Mb deletion on chromosome 12 from father • Couple pregnant again, can we diagnose it? Non-invasive microdeletion diagnosis 4.2 Mb deletion Peters et al, NEJM, 2011 FUTURE • Noninvasive fetal microdeletion detection (NOW) • Whole Exome sequencing to diagnose or rule out syndromes in utero • FETAL cells isolation from maternal blood Parental desire, prenatal whole exome sequencing (WES) Total n= 186 Agree Neutral Disagree Would want to know cause of medical problems (n= 183 170 (92.9%) 10 (5.5%) 3 (1.6%) Would do any available genetic test (n= 183) 126 (68.9%) 48 (26.2%) 9 (4.9%) Prenatal diagnosis is important (n= 183) 101 (55.2%) 60 (32.8%) 22(12%) Prenatal WES should be offered (n= 183) 152 (83.1%) 27 (14.8%) 4 (2.2%) Would want prenatal WES (n=182) 97 (53.3%) 73 (40.1%) 12 (6.6%) Would want prenatal WES even if no indication (n= 182) 63 (34.6%) 55 (30.2%) 64(35%) Kalynchuk et al, Prenatal Diagnosis, 2015 Parental opinions regarding result of WES Total n= 186 Agree Neutral Disagree Treatable childhood conditions (n= 182) 175 (96.2%) 7 (3.8%) 20 (11.0%) Non-treatable childhood conditions (n= 182) 157 (86.3%) 17(9.3%) 8(4.4%) Treatable adult-onset conditions (n= 183) 139 (76.0%) 27 (14.8%) 17 (9.3%) Treatable adult-onset conditions (n= 183) 136 (74.3%) 26 (14.2%) 21 (11.5%) Adult-onset condition would cause anxiety (n= 181) 127 (70.2%) 41 (22.7%) 13 (7.2%) VUS would cause anxiety (n=182) 130 (71.4%) 34 (18.9%) 18 (9.9%) Turnaround time is important in pregnancy Gynecology Genomics Uterine Leiomyomas • Benign tumors arising from the smooth muscle layer of the uterus • Clinically diagnosed in 25% of women • Reproductive years (20-50 years) • Tumors are monoclonal in origin • Some of the symptoms often associated with fibroids are -Pelvic pain -Complications in pregnancy -Heavy Menstrual bleeding -Infertility Human Myometrium Human Leiomyoma Tumor versus Normal • We selected 5 matched karyotypically normal leiomyomas and matching normal myometrium • High throughput sequencing with exome capture (Agilent). • Identify variants present in the tumor but not in the normal tissue Whole exome sequencing Genomic DNA 1 2 3 4 5 6 Exon Fragmentation of DNA (300-500 base pairs) Intron Capture of exon containing fragments Sequencing Bionformatics NORMAL TUMOR NextGENe software; SoftGenetics (State College, PA) Med 12 mutations in leiomyomas • In total, 148 tumors and 78 myometrium (normal) samples were screened via Sanger sequencing • No MED12 variants detected in myometrium samples • 100/148 (67.6%) tumors harbored heterozygous MED12 variants • All variants located in exon 2 or at intron 1-exon 2 junction – Missense SNVs: 79/148 (53.4%) – Deletions/Indels: 19/148 (12.8%) – Splice site SNVs: 2/148 (1.4%) Majority of SNVs in codon 44 of Exon 2 • Most common non-synonymous SNP was c.131 G>A • Glycine to Aspartic amino acid change (Non Polar to acidic) MED12 (Mediator complex subunit 12) • Transcriptional regulator complex which bridges DNA regulatory sequences to RNA polymerase II initiation complex • Total of 26 subunits • Located on the X chromosome, mutations are expressed in the tumors • Germline MED12 mutations and two forms of X-linked mental retardation: Opitz-Kaveggia syndrome and Lujan-Fryns syndrome Med12 genetics Myometrium Leiomyoma x x X X Xm X Med12 mutation, c.131G>A, in the absence of WT Med12 Mittal et al, J Clin Invest, 2015 Human and mouse leiomyomas share some common aberrations Mittal.P et al., JCI, In Press (2015) Chromothripsis Health Impacts of Ovarian Aging Hartge P., Nature Genetics 2009 Primary ovarian insufficiency clinical characteristics • Woman less than 40 with amenorrhea more than 4 months • Two serum FSH levels in menopausal range • Varying and unpredictable ovarian function 50% of cases • 5-10% of women conceive and deliver after diagnosis • In 90% of cases the cause is unknown Nelson, L., N Engl J Med 2009;360:606-14.
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