Here we demonstrated the use of the Metabochip genotyping array to identify SNPs associated with AM and ANM in a sample of African American women. Previous GWAS studies for AM and ANM have been performed in primarily European descent populations; generalization to diverse populations has largely been lacking 
. Our study is the first, to our knowledge, to consider this trait in a large African American cohort. We were able to generalize only one previously identified variant for AM and two variants for ANM to our African American cohort [AM: rs1361108; ANM: rs897798 and rs9385399 (proxy for rs1361108)]. Overall, however, we were unable to generalize the majority of significant associations for previously identified SNPs associated with AM, including LIN28B
or the 9q31 locus, or with ANM, including MCM8
, which have recently been identified in several GWAS of European-descent women. Our inability to replicate earlier findings in our African American sample may have, in part, resulted from scant Metabochip coverage of these regions. The emphasis of the Metabochip on genes involved in lipid metabolism and cardiovascular traits is evident comparing coverage in the FTO
region (1053 SNPs) to the LIN28B
region (28 SNPs).
In the discovery phase of our AM analysis, none of our results reached genome-wide significance. However, the ANM analysis yielded three associations that were significant after multiple testing corrections. Broadly, we demonstrate the ability to potentially uncover new variants associated with age at natural menopause in our African American cohort using the Metabochip.
Several studies have shown relationships between a woman’s reproductive milestones (AM, ANM, parity) and menstrual characteristics and risk for breast cancer, endometrial cancer, and ovarian cancer 
and chronic diseases such as diabetes, osteoporosis and cardiovascular disease (briefly 
). The most significant result in the ANM analysis was a SNP located upstream of LDLR
(rs189596789) which encodes a low density lipoprotein receptor implicated in familial cholesterolemia. KCNQ1
(rs79972789) also reached genome wide significance in our ANM analysis. Numerous variants in KCNQ1
have also been implicated in type 2 diabetes in several populations, though none were in linkage disequilibrium with rs79972789 
. Recently, Buber et al.
evaluated the role of menopausal hormonal changes with cardiac events in women with mutations in KCNQ1
and congenital long-QT syndrome (LQTS) 
and determined the onset of menopause was associated with an increase in the risk of cardiac events in LQTS women. Though not significant, suggestive AM associations included LPL
, which are associated with type 2 diabetes and lipid metabolism (rs1372339, rs4922116, rs1273516), and TMEM18
(rs2947411), associated with obesity and body mass index 
. These ANM associations and suggestive AM associations with genes involved in cardiovascular function, lipid metabolism, and type 2 diabetes concur with research showing later AM lowers obesity and diabetes risk while earlier ANM increases risk for cardiovascular disease, obesity and insulin resistance 
Different pathways appear to be involved in the initiation and cessation of menses. Prior GWAS and linkage studies performed in European descent or Asian populations for AM and ANM show little concordance with specific genes (reviewed in 
). Our analysis is consistent with this observation. Only PHACTR1
SNPs were suggestively significant in both our AM and ANM analyses. PHACTR1
is a phosphatase and actin regulator which has been implicated in coronary artery disease 
. Its role in menarche and menopause is yet to be determined. ARHGAP42
, a Rho GTPase activating protein, has not yet been evaluated for a role in menarche or menopause. A GWAS identified intronic ARHGAP42
rs633185 is associated with blood pressure 
, but this variant is not in strong LD with ARHGAP42
variants suggestively associated with either AM or ANM in this study. A recent study by Lu et al.
, found SNPs in both TNFSF11
significant for AM and ANM 
. SNPs genotyped on the Metabochip were in weak LD with the reported SNPs and failed to reach significance in this African American sample. Given the role that both PHACTR1
play in atherosclerosis, osteoporosis and the development of lactation glands in pregnancy, further investigation on the influence of these genes in AM and ANM is warranted 
The Metabochip was designed to be a cost-effective method of genotyping approximately 200,000 metabolic and cardiovascular SNPs and SNPs in other useful regions of the genome, such as the HLA region and the X and Y chromosomes. Overall, median SNP density on the Metabochip is approximately one SNP per 370 bases 
. This coverage appears sufficient to replicate some loci associated with both cardiovascular or metabolic traits and AM/ANM. However, we found instances of previously identified genes for AM/ANM with little/no Metabochip coverage (CYP1B1, LIN28B, ESR2, and BRSK1
) which may have impacted our results. Additionally, prior studies that identified SNPs associated with AM and ANM were performed primarily in European-descent cohorts. Though our study included over 4,000 African American women, we had limited power to identify significant associations in most previously identified loci, which may explain why we failed to detect the same associations identified in European-descent GWAS. For specific tests of association, our power was impacted by sample size and by minor allele frequencies. For example, the allele frequency for rs7861820 in this African American cohort was 0.11 compared to a higher frequency observed in HapMap CEU (0.57; Table S4
). Interestingly, we were adequately powered (>98%) to generalize the intronic LIN28B
SNP, rs314277, with AM in our sample, yet failed to find an association with this SNP or with SNPs in strong LD with it.
Metabochip performance in non-European populations was recently evaluated in a pilot study in African American PAGE participants 
. In this pilot study, Buyske et al
. demonstrated that the majority (89%) of SNPs targeted by the Metabochip passed rigorous quality control with high call rates 
. Using lipid traits as an example, Buyske et al
. demonstrated that Metabochip data can be used to replicate known GWAS-identified SNP-trait relationships. Furthermore, the pilot study demonstrated that Metabochip data can be used to fine-map GWAS-identified regions to uncover potential novel index SNPs specific to African Americans in an established locus for that trait. Fine-mapping data for AM/ANM was not included in the Metabochip content. While we were able to use the Metabochip to identify potentially novel SNP-trait relationships for AM/ANM, additional fine-mapping efforts of other loci already implicated for these traits are needed. Furthermore, additional studies in general are warranted for diverse (non-European descent) populations using Metabochip or other arrays designed for fine-mapping. Admixture in the African American population and its associated decreased LD compared to European Americans challenge identification of trait-associated SNPs. Targeted fine mapping, such as use of the Metabochip, may be more appropriate in some circumstances than GWAS to evaluate specific SNPs and regions associated with particular traits.
Although the Metabochip was designed for genotyping of cardiovascular and metabolic SNPs, this study demonstrates the feasibility of utilizing such a targeted chip to identify SNP associations with age at menarche and age at natural menopause. We identified potentially novel associations with AM/ANM at loci implicated in cardiovascular traits, obesity and cancer. This may result from pleiotropic loci or may suggest that the AM/ANM timing mechanisms influence underlying disease process. With numerous genes implicated in both metabolic and cardiovascular phenotypes and both AM and ANM, further studies will allow us to consider how specific genes may influence the reproductive lifespan in women.