Genetics of Primary Ovarian Insufficiency

Zi-Jiang Chen, M.D. Ph.D.

Shandong University,

Shanghai Jiao Tong University

China

OVARIAN CLUB VII

Genetics of Primary Ovarian Insufficiency

-New Developments and Opportunities

Outline of content

• Nomenclature and controversies

• Clinical characteristics of POI

• Genetic progress and challenges in etiology of POI

Confusion and challenge

Ovarian Insufficiency

Controversy on nomenclature

No optimal strategy for infertility

No consensus on diagnosis criteria

POI remains poorly-defined and under-researched.

Heterogeneity in etiology

Terms used for ovarian insufficiency

• Gonadal Dysgenesis

• Ovarian Dysgenesis

• Premature/early menopause

• Hypergonadotrophic hypogonadism

• Hypergonadotrophic amenorrhea

• Premature ovarian insufficiency

• Premature ovarian failure

• Primary ovarian insufficiency

Welt CK. Clin Endocrinol. 2008.

Premature ovarian failure (POF)

• Classically accepted and used term in clinics

• Life-altering diagnosis: more than

infertility and affects physical and

emotional well-being

• Emotional distress: “failure”, hopeless,

incurable

OMIM Gene

POF1 FMR1

POF2 DIAPH2(POF2A)

POF1B( POF2B )

POF3 FOXL2

POF4 BMP15

POF5 NOBOX

POF6 FIGLA

POF7 NR5A1

POF8 STAG3

POF9 HFM1

POF10 MCM8 • POF is more rigorous for nominate

causative genes in genetic research

Age<40 yrs

Primary amenorrhea

Secondary amenorrhea (>4-6 month)

FSH>40 IU/L (>2 times at least 1 month apart)

Estrogen deficiency

From POF to POI: evolution of a term

Dose not include the full spectrum of progress

Heterogeneity in etiology & phenotype

Different from natural menopause

50% intermittent ovulation

5-10% spontaneous pregnancy

Specific endpoint of ovarian function

POI

Welt CK. Clin Endocrinol. 2008

Nelson, LM. N Engl J Med. 2009.

Primary ovarian insufficiency (POI)

Normal

Normal

Regular

Normal

FSH

Fecundity

Menses

Normal

Reduced

Regular

Occult

Elevated

Reduced

Regular

Biochemical

Elevated

Reduced

Irregular/absent

Overt

• First suggested by Fuller Albright in 1942

• Continuum of the variable clinical course of ovarian ageing

• A more scientific and accurate term

• 3 stages by NIH and ASRM in 2008

Welt CK. Clin Endocrinol. 2008

2015 ESHRE POI Guideline

• Guideline development group (GDG) recommends new

diagnostic criteria

Age<40 yrs

Oligo/amenorrhea >4 months

FSH > 25 IU/l (2 occasions at least 1 month apart)

• Early identification and treatment: lower FSH cut-off

level

• Suggest application of POI in clinics and basic research

https://www.eshre.eu/guideline/POI ESHRE Guideline Group on POI, et al. Hum Reprod. 2016.

• Absence of evidence-based progress of different stages of POI

• Necessity of ART & possibility of pregnancy in patients with

FSH>25IU/l ?

• Are POI patients in biochemical stage bond to experience

ovarian failure before 40 ?

• Inclusion patients at earlier stages will increase the

heterogeneity of etiology?

• How to predict the risk of ovarian failure in earlier stages ?

Questions remained

Outline of content

• Nomenclature and Controversies


• Genetic progress and challenges in etiology of

POI

Clinical characteristics of POI from SDUIVF database

59% 25%

16%

From Irregularity to amenorrhea

<1y 1-4y ≥5y

Menarche-Amenorrhea: ~10 yrs

Irregularity-Amenorrhea: ~2 yrs; >50% experience amenorrhea in 1 year

Amenorrhea-Diagnosis: ~5 yrs

821 POI with secondary amenorrhea (FSH>40 IU/l)

14.3

22.9 24.8

29.6

0

5

10

15

20

25

30

35

40

menarche irregular menses amenorrhea diagnosis

From menarche to POI diagnosis age

From menarche to amenorrhea

Characteristics ≤5 yrs 6～10 yrs 11～15 yrs >15 yrs

P n=182 n=243 n=220 n=176

Age at diagnosis (yrs) 26.1±3.8 27.2±3.2 30.5±3.3 35.4±2.7 <0.001

FSH(mIU/ml) 73.4±23.6 78.9±28.3 79.5±29.3 74.2±24.1 0.038

E2(pg/ml) 13.6(20.3) 12(19.3) 13.74(24.8) 17.12(26.6) 0.172

Age at menarche (yrs) 14.9±1.8 14.4±1.5 13.9±1.4 13.8±1.4 <0.001

Age at amenorrhea (yrs) 17.6±2.6 22.6±2.0 26.7±1.9 33.1±2.8 <0.001

Rate of family history

(%, n) 3.3%, 6/182 5.3%, 13/243 6.8%, 15/220 11.9%, 21/176 0.008

Rate of Chromosome

abnormality (%, n)

17.7%,

25/141

11.3%,

20/177 12.7%, 21/166

5.9%,

6/101 0.05

Rate of pregnancy

history (%, n) 4.4%, 8/182

12.0%,

28/243 24.5%, 54/220 55.7%, 98/176 <0.001

Our data

From menarche to amenorrhea

Duration of menses Prevalence of familial POI Frequency of chromosomal abnormality Rate of previous pregnancy history

Outline of content

• Nomenclature and Controversies


• Genetic progress and challenges in etiology of

POI

Heterogeneous in etiology

Autoimmune

10%

Environment

10%

Iatrogenic

10%

Genetic

20-25%

Chromosomal abnormality

Gene mutation

Autoimmune disease

Anti-self antibody

Autoimmune oophoritis

Viral infection

Metabolic disorder

Smoke/toxin

Ovarian surgery

Radio/chemotherapy

Idiopathic POI: ~50% remain unclear

Genetic etiology

Cytogenetic research 1

Karyotype study

Single genes causing non-syndromic POI

Pleiotropic single gene disorders in POI

Genome-wide studies in POI (GWAS)

Whole exome sequencing (WES)

Molecular research 2

Chromosomal abnormality: a common cause

• Routine practice for genetic counseling & etiological diagnosis

• Reasonable prevalence: 10-13%

• Mainly numerical defects and

rearrangements of X

chromosome

Year Frequency Sample Population

2014 18.0 100 Tunisian

2013 10.05 179 Iranian

2012 12.1 531 Chinese

2011 10.0 269 Italian

2010 10.8 1000 Tunisian

2010 12.9 147 Dutch

2010 25.3 75 Turkish

2006 8.8 90 French

2003 12.5 104 Chinese

Prevalence of CA in different population (2003-2016)

• Identify critical region and candidate

genes at Xq (FISH, aCGH)

POF1

Xq26-q28

POF2

Xq13-q21

POF1B DACH2CHM

DIAPH2N

XF5

COL4A6

PGRMC1

XPNPEP2

FMR1

The function of most genes in ovary is not clear

Few positive mutations screened in POI patients

X numerical defects 31.7%

X rearrangeme

nts 53.3%

X-autosome translocation

15.0%

Autosomal translocation

1.6% 46, XY 4.7%

• Karyotype analysis in 531 Chinese POI: 12.1%

• Karyotype-phenotype

Primary amenorrhea

Secondary amenorrhea

Familial POI

Sporadic POI


21.4% (15/70)

10.6% (49/461) 4.3%

(2/47) 12.8%

(62/484)

P 0.01 0.09

Chen, et al. Hum Reprod. 2012.

Our data


• 45, X and 45, X/46, XX: Turner syndrome

Haploinsufficiency of pivotal genes; meiotic impairment

• 47, XXX

Meiotic disturbance; overexpression of genes escaping X-inactivation

• X deletion and X- autosome translocations

The more distal deletions exert less severe effect on stature and

reproductive function than do proximal deletions

Xq23-q27 (POF1): SA, POF

Xq13-Xq21 (POF2): PA, lack of breast development, complete ovarian

failure

Haploinsufficiency; interrupt pivotal genes; defective meiotic pairing

No autosomal region appears preferentially involved

X defects

Chen, et al. Hum Reprod. 2012.

Genetic etiology


Karyotype study

Single genes causing non-syndromic

POI





• Next generation sequencing (NGS)

• Hypothesis-driven candidate gene strategy

Function in folliculogenesis and oogenesis

POI phenotype in transgenic animal model

POI pedigree

Genome wide association study (GWAS)

Microarray-CGH

Whole exome sequencing（WES）

Custom target sequencing microarrays (TSM)

Whole genome sequencing (WGS)

Linkage analysis

Strategy to identify causative genes

Nearly 80 candidate genes identified with only 10 validated causative functionally.

Chen, et al. Hum Reprod Update. 2015.

Distribution of candidate genes

• BMP15 (Bone morphogenetic protein 15)

• AR (Androgen receptor )

• FOXO4 (Forkhead box O4)

• PGRMC1 (Progesterone receptor membrane component-1)

• DACH2

• POF1B.....

Validated functional impairment

1 392

TGF-b C terminal Signal peptide Propeptide

BMP15

H81R G199R R329C S5R

R68W R138H

L148P

A180T/F/S+V

R61W/E R76C/H N196K

R206H E211X

W221R L243G

Y235C

1 195

Cyt_B5-like_heme/steroid-bd Tansmembrane

PGRMC1

H165R P186S

Genes on X chromosome

Oogenesis/ folliculogenesis

Hormone associated

DNA damage repair/meiosis

, , GPR3,

PTEN, KITLG, SOHLH1, SOHLH2, POU5F1...

FSHβ, , Inhibins, AMH, AMHR2,

ESR1

DMC1, MSH4/5, SPO11, , HFM1,

STAG3, SYCE1

Validated functional impairment

GDF9, FIGLA, NOBOX, NR5A1, NANOS3

FSHR, LHCGR

MCM8/9

Genes on autosomes

Genes Mutation rate References Genes Mutation rate References

EXO1 Wang, 2016 FST Liu, 2012

WT1 0.5% Wang, 2015 TGFBR3 1.8% Qin, 2011, 2012

HELQ Wang, 2015 WNT4 Chen, 2011

PTEN Zou, 2015; Chen, 2014 POU5F1 0.9% Wang, 2011

SOHLH1 0.36% Zhao, 2014 FOXO3 13.2% Wang, 2010

SOHLH2 1.4% Jiao, 2014 GPR3 Zhou, 2010

PGRMC1 0.5% Wang, 2014 FOXL2 Ni, 2010

NR5A1 0.26% Jiao, 2014 GDF9

Wang, 2010

DMC1 Wang, 2014 1.0% Zhao, 2007

NANOS1 Wu, 2013 NOBOX Qin, 2009

NANOS2 Wu, 2013 SALL4 2.0% Wang, 2009

CDKN1B Zhao, 2013; Wang, 2010 FIGLA 2.0% Zhao, 2008

NANOS3 1.0% Wu, 2013. FSHR Chen, 2006

Qin, 2007

Candidate genes screening in Chinese POI patients

Gene Location Mutation

rate Amino acid

change Mechanism Reference

PGRMC1 Xq22-24 0.5 p.P186S Wang, et al., 2014 FMR1 Xq27 0.52 Guo. et al. 2014

FIGLA 2p13.3 4 p.A4E No effect

Zhao, et al., 2008 p.G6fsX66 Prematurely terminate translation p.140delN Impaired interaction with TCF3

FSHR 2p16 1 p.M265V

unpublished p.A59* Truncated protein

FAM175A 4q21 0.5 p.L243V unpublished

MSH5 6p21.3 1.6 p. L353M p. D487Y p. I703V

Impaired DNA repair for DSB unpublished

NR5A1 9q33 0.26 p.Y5D Impaired transactivation on Amh, Inhibin-a, Cyp11a1, Cyp19a1

Jiao, et al., 2014.

SOHLH1 9q34.3 0.55 p.S317F; p.E376K

Zhao, et al., 2015

WT1 11p13 0.5 p.P126S; p.R370H

Impaired transcription on downstream genes. Wang, et al.,2015

CSB-PGBD3 0.23 p.E215X; p.V1056I;

Impaired DNA repair Qin. et al. 2015

SOHLH2 13q13.3 1.1 p.E79K;

p.E105G; p.T321P

Qin. et al. 2014

MCM8 20p12.3 1.04 p.H317L; p.H601R

Impaired DNA repair unpublished

Causative genes identified in POI patients from SDUIVF

Our data

Genes in oogenesis/ folliculogenesis

Toxin...

Normal ovary

POI POF

Genes in meiosis/DNA damage repair

Premature failure

Genes in hormone synthesis/effect

Pathogenesis

• More candidates, few causative

• Notable ethnic differences in mutation frequency

• Not only ovarian exclusively or preferentially, but also expressed

ubiquitously

• Many genes that appear isolated actually may be interrelated

functionally within yet to be defined pathways

• New identified genes focus on meiosis and DNA damage repair

mainly by WES in POI pedigree

• The pathogenesis, association with phenotype and risk

prediction still needs to be explored

Current status of genes causing POI

Genetic etiology


Karyotype study

Single genes causing non-syndromic

POI





FMR1: Fragile X syndrome

FOXL2: Blepharophimosis-ptosis-epicanthus syndrome (BPES)

NOG: Proximal symphalangism (SYM1) and Multiple synostoses syndrome

(SYNS1)

GALT: Galactosemia

PMM2: Carbohydrate-deficient glycoprotein (CDG) deficiency

AIRE: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy

(APECED)

EIF2B: Ovarian leukodystrophy

HSB17B4, C10orf2, HARS2, LARS2 and CLPP: Perrault Syndrome (PS)

POLG: Progressive External Ophthalmoplegia (PEO) Majority has no positive mutations in sporadic non-syndromic POI patients


• Different CGG repeats are associated with diminished ovarian

function

• Ethnic heterogeneity in FMR1 premutation prevalence in POI

population

Caucasians: sporadic (3.3-6.7%) familial ( 13%)

Chinese: sporadic (0.5%)

Chen, et al. PloS ONE. 2015.

FMR1 and POI

FMR1 premutation is an uncommon explanation for POI in Han Chinese

Our data

Genetic etiology


Karyotype study






Sigle gene analysis GWAS/WES

• Single gene sequencing: mutation frequency mostly <

5%; based on functional hypothesis

• Genome wide approaches: GWAS, array-CGH, WES,

efficiently locate susceptible loci or genes; identity

disease associated signaling pathway

Genome wide approaches

Kang et al., 2008

Knauff et al., 2009 Qin et al., 2012 Pyun et al., 2012 Oldenburg et al., 2008

Caburet et al., 2012

Ethnicity Korean Caucasian Dutch Chinese Korean Dutch Middle-Eastern

Discovery Set

No. cases 24 99 391 24 10 5

No. controls 24 181 895 24 5 4

Association PTHB1 Rs246246 in ADAMTS19 intron

8q22.3 22 SNPs in LAMC1 5q14.1-q15 7p21.1-15.3, 7q21.3-22.2

Replication Set

No. cases 101 60 400 98 - -

No. controls 87 90 800 218 - -

Results PTHB1 is associated with POF

Association not confirmed

8q22.3 is associated with POF

High frequency of 9 SNPs and 1 haplotype in POF

- No causal mutations In candidate genes DLX5, DLX6, DSS1

References Review

GWAS

Ethnicity Cases CNVs Potential candidate genes

Aboura et al., 2009

French 99 8 known DNAH5, NAIP, DUSP22, NUPR1, and AKT1

Ledig et al., 2010

German 44 24 CNVs -

McGuire et al., 2011

Caucasian 89 17 novel microduplications and 7 microdeletions

2 novel microdelets with haploinsufficiency of SYCE1 and CPEB1

Knauff et al., 2011

Dutch Caucasian

108 One CNV in Xq21.3 PCHCHIIX and TGHF2LX

Norling et al., 2014

Swede 11 11 3 NOBOX-binding elements and an E-box for GDF9

Quilter et al., 2010

UK 42 15 novel discrete X chromosome intervals

-

Dudding et al.,2010

New Zealand

50 2 microduplications (Xp22.33 and Xq13.3)

-

References Review

CNVs in array CGH studies

Chen, et al. Hum Mol Genet. 2012.

Our data

• GWAS

Sporadic POI: 791cases

Control: 1695 cases

• New susceptible region: 8q22.3

P=8.5×10-8, OR=0.7

An important yet undefined

long-distance regulatory

region might affect ovarian

differentiation and oogenesis

Susceptible 8q22.3 associated with Chinese POI

• Multiple susceptible loci potentially associated

with POI were identified in different populations

• Remain difficult to isolate the new causative genes

(significance<10-6)

• Limited statistical power to detect a reliable association

• Few positive results have been replicated

• Low prevalence of POI and high heterogeneity in

different populations/patients

Challenges of GWAS

Genetic etiology


Karyotype study






Gene Location Mutation Genotype References

HFM1 1p22.2 c.1686-1G>C; p.I884S Compound het

Wang et al., 2014 p.G736S; p.P1310R fs*41 Compound het

MCM9 6q22.31

c.1732+2T>C Homo Wood-Trageser et al.,

2014 p.R132* Homo

p.E495* Homo Fauchereau et al., 2016

STAG3 7q22.1

p.F187fs*7 Homo Caburet et al., 2014

p.Y650Sfs*22 Homo Le Quesne Stabej et al.,

2016

SYCE1 10q26.3 p.Q205* Homo De Vries et al., 2014

MCM8 20p12.3 p.P149R Homo AlAsiri et al., 2015

eIF4ENIF1 22q11.2 p.S429* Het Kasippillai et al., 2013

WES in non-syndromic POI pedigree References Review

Familial POI: p.G278D (PGBD3)

Sporadic POI: p.V588I (PGBD3), p.E214X (CSB)

CSB-PGBD3 is expressed in the nuclei of oocytes

IHC

ISH

WES in POI pedigree-1

Sanger sequencing of CSB-PGBD3 in 432 sporadic POI patients

Chen, et al. Plos Genet. 2015.

CSB-PGBD3 mutations in a Chinese POI pedigree

Our data

Chen, et al. Plos Genet. 2015.

CSB-PGBD3 mutations in a Chinese POI pedigree

Our data

The underlying mechanism−inability to repair DNA

damage−is plausible and introduces a concept for POI

• Efficient strategy to identity causative genes by combination of

linkage analysis and WES

• Almost all genes identified by WES involve in meiosis and DNA

damage repair

• Perturbation of gene or signaling pathway in meiosis might result in

autosomal recessive POI in women

• Causative function of genes/pathway related to meiosis and DNA

damage repair in sporadic POI needs to be further explored

Current status of WES

• NGS: 12 non-syndromic POI

• Coding regions of 70

candidate genes were

massively sequenced

• Mutations in ADAMTS，

BMPR2 and LHCGR were

identified

Fonseca, et al. Fertil Steril. 2015.

Next generation sequencing (NGS)

• Most studies so far largely focus on coding variants

Non-coding RNA (miRNA, lnRNA...)

Disrupt/create alternative splicing or transcription factor-binding sites

Epigenetic modifications (DNA methylation, chromatin modification)

• Variants within introns or inter-gene region

• Regulatory genes and network: RNA-seq/Methyl-seq/Chip-seq will

allow systematic study of the transcriptome in ovaries in relation to risk

genotypes that have been shown to contribute to POI

Future direction

• In-vitro activation (IVA) +IVF-ET:

Li, et al. PNAS, 2011. Kawamura, et al. PNAS, 2013. Suzuki et al. Hum Reprod, 2015.

Treatment with PI3K activators stimulates

dormant primordial follicles by PI3K-AKT-FOXO3

pathway

Ovarian fragmentation promotes secondary

follicle growth by Hippo signaling pathway

• Gene editing in familial

POI？ CRISPR/Cas9

technique？

Translational medicine in POI

Highly heterogeneity in phenotype and causes

Endogenous genetic defect plus exogenous damage

Monogenic disorder caused by different mutations in different genes

Heterogeneity in different population, even in different individuals

with same ethnicity

Future direction

Genes in meiosis and DNA damage repair

Non-coding genes and RNA

Interaction between genetics and environment

Epigenetics

Evidence based longitudinal clinical research to monitor disease

progress

Take home message

Zi-Jiang Chen’s Group Institute of Zoology, Chinese Academy of Sciences, China

Fei Gao Tieshan Tang

Joe Leigh Simpson Aleksandar Rajkovic

University Hospitals of Leicester, United Kingdom

Farook Al-Azzawi

Raymond Dalgleish

Acknowledgement

Documents

Genetics of Primary Ovarian Insufficiency