Genetics of Menopause Timing: Findings from the ReproGen Consortium
MaryFran Sowers Memorial LectureApril 3, 2018
Joanne M. Murabito MD ScM
Associate Professor of Medicine
Boston University School of Medicine
Presentation Overview
• Relation of age at menopause to later life health
• Genetic association studies of age at natural menopause
• Follow-up analyses to understand the biologic relevance of the genetic associations & the link to health
http://rinkwestmedical.co.za/wp-content/uploads/2013/08/Womens-health-640x350.jpg
Can Age At Natural Menopause (ANM) as a phenotype uncover important biology?
“the genetic associations…. should provide insight into timing of menopause
but also may yield needed breakthroughs in understanding the genetic
variation conferring risk of breast cancer, cardiovascular disease and other
health events of later adulthood” Patricia Hartge, NCI Nature Genetics 2009
CVD Mortality Trends: US Men and Women
Mehta LS Circ 2016 Acute MI in Women
1 in 3 deaths in women attributed to CVD
Benjamin EJ et al Heart Disease and Stroke Statistics 2018 Update
Women Aged <45 Years at Onset of Menopause vs Women >45 Years at Onset
Muka T et al 2016 JAMA Cardiol
Risk of Mortality According to Menopause Timing
Coronary Disease Stroke
Wellons M Menopause 2012;19:1081
Menopause Timing & Cardiovascular Disease
Multi-Ethnic Study of Atherosclerosis
Early Menopause Early Menopause
Rates of CVD and Breast Cancer in Women by Race/Ethnicity
Mehta LS, et al,2018 Circ AHA Statement: CVD and Breast cancer
Menopause linked to epigenetic age
• DNA methylation age or epigenetic age is biomarker of human aging
• Predicts mortality; potential risk marker CVD
• Age at menopause associated with epigenetic age• Older epigenetic age associated with younger age at menopause
• GWAS of two epigenetic age traits recently published• Age at menopause genetically correlated with epigenetic age
• Each one year earlier age at menopause associated with 0.4 year higher intrinsic epigenetic age (p=3.5 x 10-3)
Levine ME et al 2016 PNAS; Lu AT et al 2018 Nat Comm; Roetker NS, et al 2018 Circ Genom Precis Med
Genetics of Menopause Timing• Identify genetic variants associated with menopause timing
• Provide mechanistic insights into ovarian aging
• Improve understanding of how menopause timing influences later life health
Menopause Age Has High Familial Concordance & Heritability
• Women with mother or sister with early menopause (< 45 years) had 6 fold increase in early menopause
• Women with mother or sister with late menopause (> 54 years) at increased risk late menopause
• Heritability of ANM ~ 50% → 50% of the variation in ANM is due to genetic factors
• Variation in ANM explained by smoking, parity, SES, decade of birth only 5%
Morris DH: 2011 Menopause 18:956
Age At Natural Menopause Trait Definition
• Have your periods stopped for one year or more?
• Age period stop?
• Cause periods stop→ natural
• Menopause normally occurs between 40 to 60 years Reported Age at Natural Menopause:
Framingham Heart Study Women
0
5
10
15
20
25
18 22 26 30 34 38 42 46 50 54 58 62
Age (years)
Perc
ent (%
)
POI
Murabito, et al 2005 JCEM
Self-report and prospective age at menopause are highly concordant
Comparison of self-reported data
• NSHD recruited at birth followed regular intervals
• Million Women Study recruited in middle age
• 541 women in both studies of similar age
• Compared self-reported age at menopause in the two studies
• Mean age 48 years
Cairns BJ et al 2011 BMC Med Res Methodol
ReproGen Consortium: Menopause
• International: US, Europe, Australia
• GWAS of age at natural menopause (ANM)
• Women of European ancestry
• 22 studies in the discovery stage, n=38,968
• 21 studies in the replication stage, n=14,435
• Age at natural menopause between 40 - 60 years• Mean age at natural menopause 48.4 to 50.8 across studies
• Excluded surgical menopause, chemo/radiation, women using HRT or OCP
Stolk, et al Nature Genetics 2012; http://www.reprogen.org/index.html
RHBDL2
EXO1TLK1111 HELQ
TRD3
UIMC1
MPPED2
PRIM1
KPNA3
POLG
NLRP11
Meta-analysis Results for Menopause: Highlight DNA repair & Immune pathways
Stolk L et al NG 2012; Stolk L,et al NG 2009; He C, et al NG 2009
MCM8
TMEM150B, BRSK1
SYCP2L
PRRC2A
ASH2L
FNDC4
In LD with SNPs in FSHBIn LD with SNPs in FSHBIn LD with SNPs in FSHBIn LD with SNPs in FSHB
• MCM8 mutations linked to autosomal recessive disorder: primary amenorrhea, hypothyroidism,
hypergonadotropic hypogonadism
• MCM8 complexes with MCM9 to function in homologous recombination (HR) mediated DNA repair
AlAsiri S et al 2014 J Clin Invest; Bouali N 2017 Fertilty Sterility; Desai S 2017 J Clin Endo Metab
Expanded Menopause GWAS & Exomechip Investigation
• Common and low-frequency protein-coding variation
• 33 studies, ≈69,000 women
• Exomechip genotyping, 22 studies, n=39,000
• Follow-up analyses�Pathway analysis
�Mendelian Randomization
HapMap & Exomechip SNP Associations for Menopause
HELB
SLCO4A1
54 independent SNPs in 44 genomic loci2 rare coding variants
Day FR, et al 2015 Nature Genetics
Common & exome chip variants at SLCO4A1 appeared independent
Day FR, et al. 2015 NG
Multiple Signals at HELB
(DNA helicase B)Top Exomechip signal
maps to an acidic motif & results in
replacement of an aspartate by a
nonpolar glycine residue affects
binding of the helicase.
Biologic Pathway analyses MAGENTA & GRAIL
• We also tested for signal enrichment using pre-identified biologic pathways using all SNPs in the GWAS
• Custom pathways• Primary ovarian insufficiency
• Ovarian function
• Monogenic disorders of puberty
ANM SNPs strongly enriched DNA damage response (DDR) pathways
Adapted from Jackson & Bartek Nature 2009
29/44
regions
contain DDR
gene (s)
STRING Network Analysis:
BRCA1 linked to 15 genes from Menopause GWAS
Common variant in BRCA1 associated with ANM
http:// string-db.org Search Tool for Retrieval of Interacting Genes
Potential Mechanism Linking DDR pathway to Menopause
Oktay K et al 2014 Am Soc Clin OncOktay K et al 2014 Am Soc Clin Onc
ANM SNPs enriched for Primary Ovarian Insufficiency Genes
EIF2B4
MSH5
TDRD3
POLG
MCM8
Day FR, et al 2015 Nature Genetics
Significant enrichment of all ANM SNPs with monogenic puberty gene set (p=0.01)
• TAC3
• CHD7
• KISS1R
• FGFR1
• SOX10
� Animal models show signaling in ovary
(TAC3, KISS1R) in addition to
hypothalamus
� Expression TAC3 and KISS1 in human
female reproductive tissues
� May indicate a neural influence on the
timing of ovarian follicular aging
5 menopause SNPs in/near genes for hypogonadotropic hypogonadism
Is there a link between ANM variants and breast cancer?
• Known epidemiologic link between menopause age and breast cancer risk
• We observed an enrichment with DNA damage response genes
• Would risk be related to this pathway?
• Collaboration with Breast Cancer Association Consortium (BCAC)
• Across the 56 ANM-SNPs there was a positive correlation between effect sizes in ANM and the effect sizes for breast cancer risk in the BCAC
Test causal relation between ANM & breast cancer using Mendelian Randomization
Robinson PC, et al. Nature Reviews Rheum 2016
Use genetic risk scores as the score explains a larger proportion of the
variance in the exposure than any single variant
Mendelian Randomization
• Polygenic Risk Score (PRS):• 56 ANM SNPs (54 HapMap + 2 Exome)
• Weighted by each SNP’s effect size on menopause
• > 40,000 breast cancer cases & >40,000 controls from BCAC
• Tested association between PRS and breast cancer using logistic regression
• 1-unit increase in PRS = 1 year increase in genetically predicted age at natural menopause
PRS and Breast Cancer Risk:Genetically predicted 1-year increase ANM
Odds Ratio
Confidence Interval
P-value
All SNPs 1.06 1.05, 1.08 2.78 x 10-14
OR=1.030 (1.026-1.034)Largest pooled analysis epidemiological studies
Odds Ratio
Confidence Interval
P-value
38 DDR SNPs 1.05 1.03, 1.08 1.06 x 10-7
17 Non-DDR SNPs 1.12 1.06, 1.21 7.84x 10-10
P het= 0.01
Quintiles of menopause PRS
ER positiveOR=1.07 (1.05-1.10)
P=1.73x10-12
ER negativeOR=1.03 (1.00-1.07)
P=0.04
Genetically predicted ANM had a larger effect on ER-positive than ER-negative Breast Cancer
Bre
as
t C
an
ce
r O
R
ANM Genetic Risk Score and First CVD Event
Cohort HR 95% CI p-value
FHS 1.22 1.08, 1.37 0.001
ARIC 1.06 0.93, 1.22 0.36
RSI 1.06 0.99, 1.14 0.11
RSII 1.27 0.95, 1.69 0.11
Meta-analysis 1.10 1.04, 1.16 9.7 x 10-4
Adjusted for age, PCs, family relatedness, study center and CVD
risk factors
1-unit increase in GRS corresponds to a 1 year decrease in ANM
Sarnowski C 2017 Menopause
Genetic Correlation between ANM & GWAS traits
Phenotype Reg. Coef. SE P-value
CAD -0.22 0.09 0.011
BMI -0.13 0.04 0.002
Weight -0.12 0.04 0.003
Waist circumference -0.12 0.05 0.018
Hip circumference -0.12 0.04 0.001
T2DM -0.06 0.07 0.42
Women
Sarnowski C, et al 2017 Menopause
FSHB ANM SNPAssociated with Other Reproductive Traits
Trait SNP Direction Study
ANM rs11031006 Earlier ANM Day FR 2015
Menstrual cycle length rs564036233 increase Ruth KS, 2016
PCOS rs11031006 Day FR 2015
LH in PCOS rs11031006 Increase levels Hayes 2015
Dizygotic twinning rs11031006 Mbarek 2016
Endometriosis rs74485684 Sapkota 2017
http://www.ebi.ac.uk/gwas/search?query=FSHB
1000G Meta-analysis for Menopause in progress
• Will include full UKBiobank sample
• Preliminary results: more than doubled the number of signals
• Continue to see signals in DDR pathway
• Gene x environment interactions
• Mendelian Randomization to examine additional disease links such as osteoporosis
Are loci influencing menopause in White women important in women of other ancestries?
• Few GWAS of menopause conducted in women of diverse backgrounds
• Race/ethnic diversity in genetic background
• Risk for many important health outcomes
• Distinct reproductive profiles
• Genetic architecture underlying reproductive aging may differ across ancestry groups
GWAS ANM in African American Women
Collaboration across 11 U.S. studies
N=6510 women
• CARe: ARIC, JHS, MESA,CHS
• WHI
• AA Breast Cancer Consortium
• GENOA, HABC, MESA family
• Mean ANM: 48.8 to 50.2 years
No genome-wide significant associations
Inter-genic SNP on chr 17 lowest p=1.6 x 10-6
Chen C. et al 2014 HMG
Replication of ANM loci identified in EA women in African American Women
• Evaluated SNPs in 30 loci
• Many loci were associated in AA women, directionally consistent
• Of note, the MCM8 SNP in EA women with the largest effect size did not replicate in AA women→ uncovered a second signal
• MAF differences (7% EA women vs. 1.3% in AA women)
• Effect size differences (1.07 EA women vs 0.35 AA women)
What about women of other ancestries?
• Data sparse, small samples, limited number of SNPs/genes tested
• Population Architecture using Genomics & Epidemiology Study
• 5 ANM SNPs genotyped in American Indians, AA, Asians, Hispanics, Native Hawaiians
• Replicated 3 SNPs, most robust for MCM8 SNP:
Carty et al 2013 Hum Reprod
rs16991615 N CAF Beta P-value
EA 10955 0.07 0.82 7.8 x 10-15
AA 2129 0.02 0.57 0.22
Asian 1978 0.0001 1.25 0.46
Hispanic 1143 0.06 1.05 0.0004
Am Indian 934 0.02 2.63 0.001
N Hawaiian 294 0.03 -0.41 0.60
Trans-ethnic meta-analysis in progressAA women: n=5710 menopause, n=14488 menarche
Hispanic women: n=3706 menopause, n=10661 menarche
Asian women: n=1838 menopause, n=4350 menarche
Ovarian Aging Study: Markers of Ovarian Reserve
• Community-based cohort of women recruited from eligible KP members
• Evaluated markers of ovarian reserve• FSH
• AMH
• Antral follicle count
• No genome-wide significant results
• MCM8 SNP associated with later ANM, associated with higher follicle count (p=0.018)
Schuh-Huerta SM, et al Hum Reprod 2012; Schuh-Huerta SM et al Hum Genet 2012
Conclusions and Future Directions
• Genetic association studies have identified variants for menopause timing linked to later life health conditions
• Whole Genome Sequencing leveraging TOPMed resource
• Further studies in women of diverse ancestry are needed• New genetic variants in non-European populations, using better
genome coverage of rare variants specific to each population
• Functional studies are needed to identify causal variants and to further our understanding of the biologic mechanisms of menopause timing and links to health outcomes
ReproGen Consortium Investigators
Felix DayKatherine RuthDeborah ThompsonKathryn LunettaNatalia PervjakovaDaniel ChasmanLisette StolkHilary FinucanePatrick SulemBrendan Bulik-SullivanTonu EskoCathy ElksAndrew JohnsonKari StefanssonJenny Visser Ken OngJenny Chang-ClaudeJohn RB PerryAnna Murray
FundingAG029451AG032598
Women Aged 45-49 Years at Onset of
Menopause vs Women >50 Years at Onset Women Aged <45 Years at Onset of
Menopause vs Women >45 Years at Onset
Muka T et al 2016 JAMA Cardiol
Risk of Mortality According to Menopause Timing
Women Aged <45 Years at Onset of Menopause vs Women >45 Years at Onset
Muka T et al 2016 JAMA Cardiol
Risk of CVD Mortality According to Menopause Timing
Menopause & Type 2 Diabetes
• Epic-InterAct study
• Country specific hazard ratios of type 2 diabetes per standard deviation decrease in menopausal age
Brand JS 2012 Diabetes Care