Genomic Approaches to Reproductive Disorders...2015/10/27 · Fragile- X screen
Transcript of Genomic Approaches to Reproductive Disorders...2015/10/27 · Fragile- X screen
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