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 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 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 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 ( 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 (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. Clinical Evaluation of POI

• Physical Examination • Serum Prolactin • Thyrotropin • FSH levels • Karyotype (Turner, mosaic, X/autosome translocations, XY sex reversal) • FMR1 (1-2% of sporadic, 5-13% familial)

Rebar, RW. Obstet and Gyn, 2009 Focus on women likely to have genetic cause

Primary amenorrhea

Secondary amenorrhea <25 years of age

Familial ovarian failure

Which nucleotides are pathogenic?

Frequency in the population, common variants are >5%

Is mode of inheritance satisfied (recessive, dominant, de novo, sex)

What kind of a mutation is it? (Frame shift, stop codon, splicing site)

Is the mutation conserved?

Is the expressed in the ovary?

Is there an animal model that proves its importance in ovaries? Family with POI

1 2 3 I 1 2 Female II Male 1 2 3 4 ADPKD III Heterozygous parent 1 2 Affected IV daughter Deceased EPL 1 2 V 3

Katari et al, JCEM, 2015 Clinical Data

Table 1: Laboratory Profile of Affected Daughters (V-1 & V-2)

Normal Rangea V-1 V-2

FSH mIU/ml 1-9.2b 87 104.6

LH mIU/ml 0.3- 29.4b 37.1 33.3

Estradiol pg/ml 30-300c 22 20

TSH mIU/ml 0.3-5c 1.6 0.59

Free T4 ng/dl 0.75-1.54c 0.9 1.09 Adrenal antibody Negative Negative Negative screen

Karyotype 46 XX 46 XX 46 XX

Fragile- X screen <44 CGG repeats Negative Negative aAll hormone measures provided were prior to hormone replacement therapy. bReference range for adolescent girls between ages 12-14. c Mayo Clinic, Mayo Medical Laboratories, Mayo Clinic, Minnesota. Pedigree Sanger Sequencing 1 2 3 I 1 2 WT/MT MT/MT II C A T/G T C A G T 1 2 3 4 III 1 2 IV

1 2 V 3

Mutation in FSHR, chr2:49,190,707 c.1253T>G, p.Ile418Ser Multiple affected women with POI Daughters with Premature Ovarian Insufficiency MCM8 mutation

AlAsiri S, et al. J Clin Invest. 2015; 125:258-62. MCM9 mutation

Wood-Trageser MA, et al. Am J Hum Genet. 2014; 95:754-62. Mcm8-/- and Mcm9-/- female mice are sterile Mcm8-/- and Mcm9-/- Ovaries: 1. Atrophied 2. Dysplastic primary follicles 3. Infertility Mcm8-/- Testes: 1. Azoospermia, meiotic I prophase block Mcm9-/- Testes: 1. Small, some tubules have spermatozoa, fertile

Hartford SA, et al. Proc Natl Acad Sci U S A. 2011;108:17702-7, Lutzman, et al. (2012) Molecular Cell MCM8 and MCM9 deficiency Causes Chromosomal Breaks

Lutzman, et al. (2012) Molecular Cell MCM8 and MCM9 are DNA repair involved in HR

M M C C M M 8 9

Sasaki, Lange, Keeney, Nature Reviews, 2010 Cells from Patients with MCM8 c. 446C>G Mutation Display Chromosomal Instability

A

B MCM8 and MCM9 mechanism of action

M M C C M M 8 9

Germ cell Apoptosis Primordial cluster Follicle

M C M 9 Chromosomal instability syndromes associated with POF

SYNDROME GENE • Fanconi Anemia FANCA • Ataxia-telangiectasia ATM • Bloom’s syndrome BLM • Werner syndrome (Rothmund-Thompson) WRN • New Syndrome MCM8,MCM9 Loci involved in DNA break repair associate with age of menopause

• In GWAS studies, non-synonymous MCM8 SNP shows strongest association signal with the age of menopause

• Meta-analysis of GWAS studies shows a preponderance of loci involved in DNA break repair: EXO1, BRSK1, HELQ, TLK1, SYCP2L, ASH2, UIMC1, HELB, FBXO18,MSH5,DMC1 Family #5 with idiopathic POF 58 59

Total number of reads 122,004,654 148,327,568 Exome coverage 113X 150X 55 56 SNPs, DEL, INS 88,371 76,568 Shared homozygous 9537/1178 57 58 59 Shared heterozygous 8935/1231 Exclude known SNPs, 14 years 15 years 9 Non-deleterious SNPs Inherited from each parent, AR NUP107 Disrupts Drosophila oogenesis ABCD2

Expressed in ovary NUP107 ABCD2 Animal models needed to resolve causality

ABCD2 null ABCD2 wild type Nup107 chr12:691909500 (1395 C>T, p.355R>C) females are infertile

1. Homozygous females are infertile

2. Homozygous males are fertile, 8 pups per litter

3. Wild type controls exposed to injections are fertile with 8 pups per litter Dcaf17 mutations and phenotyping Dcaf17 mutations and phenotyping POI genetics

Familial POF, we made diagnosis in 25/115 cases examined (20%)

15% of the individuals had disruptive homozygous mutations in known genes. 30% of those genes involved in meiosis.

Double strand break sensitive assays and POF, will they identify Women at higher risk for aging?

Gonadal failure is a highly heterogenous disorder, a major gene or genes are unlikely. It is also a developmental disorder. Exome Aggregate Consortium

60,706 Exomes sequenced from various populations

10% of our variants present in this consortium  meiotic meiotic arrest (3/14)and 21% azoospermic 1.6% (4/240) Mutations in Azoospermia, X Chromosome TEX11 Array - 2 2 - 1 0 1 1 0 1 P67

70045530 69954488 and X - 2 2 Array - 1 0 1 1 0 1 P89 Yatsenkoet al, NEJM,2015 - HR CGH

70046552 69954488 TEX11

Exon 10 11 12 Incidental Findings

ACMGG- American college of Medical Genetics and Genomics

Report mutations in 56 genes that are “actionable”

115 Exomes Sequenced: POF individuals and family members

RYR2 (Ventricular Tachycardia): 2 individuals

MYBPC (Dilated Cardiomyopathy): 1 individual

Approximately 1-3% of samples will end up with reportable incidental findings. Acknowledgment

Svetlana Yatsenko Tianjiao Chu

Michelle Woods Alex Yatsenko

David Peters Huaiyang Jiang

Allen Hogge Priya MIttal

Neil Devereux Yong-hyun Shing

Urvashi Surti Kemal Topaloglu