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)

Number of golden stars

none

No submitter provided an interpretation with assertion criteria (no assertion criteria provided), or no interpretation was provided (no assertion

provided)

one

At least one submitter provided an interpretation with assertion criteria (criteria provided, single

submitter) or multiple submitters provided assertion criteria but there are conflicting

interpretations in which case the independent values are enumerated for clinical significance (criteria provided, conflicting interpretations)

twoTwo or more submitters providing assertion

criteria provided the same interpretation (criteria provided, multiple submitters, no conflicts)

three reviewed by expert panel

four practice guideline

ClinVar Stars and their interpretation

De novo genomic events

• Meiotic errors (Aneuploidies)

• Mitotic errors (Mosaicisms)

• De novo deletions or duplications (DiGeorgeSyndrome)

• De novo mutations (Tuberous sclerosis, Neurofibromatosis)

Hassold and Hunt, Nat Rev Genet, 2001

Maternal age effects on incidence of trisomies

Chaos in the Embryo

Vanneste et al, Nature Medicine, 2009

PGS at UPMC

Precursor fetal cells

Embryo

Embryo biopsy Microarray PGS

Array-CGH

Precursor

placenta cells

DNA, extraction,

amplification

Simultaneously tests for

all 24 chromosomes

Male

Male

Male

Female

Embryo 1: Male -Normal

Embryo 2: Female - Trisomy 21

Embryo 3: Male – Monosomy 11

Embryo 4: Male Monosomy 15

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

2:2 inter-chromosomal tag ratio

Chromosome 21

Chromosome 1,2,3…..20,22 Chromosome 21

Normal

Trisomy 21

2:3 inter-chromosomal tag ratio

General Principle of Non-Invasive Detection of Trisomy By Shotgun

Sequencing

Chromosome 1,2,3…..20,22

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

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

Importance of Fetal Fraction

•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.

Current Indication: High Risk

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 SubchromosomalAbnormalities Noninvasively?

G banding > 4 Mb FISH 40 to 250 kb /clone

Genomic resolution 1 base pair (bp) to 10 megabase (Mb)

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

Abnormal

20 (11.3%)

Unclear

significance

16 (9%)

Incidental

findings

4 (2.3%)

Cultural

artifacts

2 (1.1%)

Normal

135 (76.3%)

Abnormal

karyotype

10 (5.6%)

Cultural

artifacts

1 (0.6%)

Normal

Karyotype

165 (93.2%)

Abnormal

Karyotype

1 (0.6%)

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

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%)

Parental desire, prenatal whole exome sequencing (WES)

Kalynchuk et al, Prenatal Diagnosis, 2015

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%)

Parental opinions regarding result of WES

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

Genomic DNA

21 3 4 5 6

Exon

IntronFragmentation of DNA (300-500 base pairs)

Capture of exon containing fragments

Sequencing

Bionformatics

Whole exome sequencing

NORMAL TUMOR

NextGENe software; SoftGenetics (State College, PA)

• 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%)

Med 12 mutations in leiomyomas

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-Frynssyndrome

Med12 genetics

Myometrium Leiomyoma

X X Xm X

x 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 gene expressed in the ovary?

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

Female

Male

ADPKD

Heterozygous

parent

Deceased

EPL

Affected

daughter

I

II

III

IV

V

1

2

3

1

2

1

2

3

4

1

2

2

13

Family with POI

Katari et al, JCEM, 2015

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

screenNegative Negative Negative

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.

Clinical Data

C A T C G TA

WT/MT MT/MT

T/G

Mutation in FSHR, chr2:49,190,707

c.1253T>G, p.Ile418Ser

I

II

III

IV

V

1

2

3

1

2

1

2

3

4

1

2

2

13

Pedigree Sanger Sequencing

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.

Lutzman, et al. (2012) Molecular Cell

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,

MCM8 and MCM9 deficiency Causes

Chromosomal Breaks

Lutzman, et al. (2012) Molecular Cell

MCM8 and MCM9 are DNA repair proteins

involved in HR

M

C

M

8

M

C

M

9

Sasaki, Lange, Keeney, Nature Reviews, 2010

Cells from Patients with MCM8 c. 446C>G

Mutation Display Chromosomal Instability

A

B

Primordial

Follicle

MCM8 and MCM9 mechanism of action

Germ cell

cluster

Apoptosis

M

C

M

8

M

C

M

9

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 SNPshows 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

14 years 15 years

Total number of reads 122,004,654 148,327,568

Exome coverage 113X 150X

SNPs, DEL, INS 88,371 76,568

58 59

Shared homozygous 9537/1178

Shared heterozygous 8935/1231

Exclude known SNPs,

Non-deleterious SNPs 9

Inherited from each parent, AR NUP107

ABCD2

55 56

5957 58

Disrupts Drosophila oogenesis

Expressed in ovary

NUP107 ABCD2

ABCD2 null ABCD2 wild type

Animal models needed to resolve causality

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

Ex

on

10

11

12

-2 -1 0 1

Array P67 Array P89

-2 -1 0 1

6995

4488

7004

6552

6995

4488

7004

5530

TEX11X Chromosome

Azoospermia, TEX11 and X-HR CGH

Mutations in

1.6% (4/240)

azoospermic

and 21% (3/14)

meiotic arrest

Yatsenko et al, NEJM, 2015

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