Urinary DENND1A.V2 as a Predictor of Pubertal Hyperandrogenemia

To date, the mechanisms underlying excess androgen production from steroid producing tissues have been unclear, but recent Genome Wide Association Studies (GWAS) have provided additional data in this regard. Initial GWAS data in a Han Chinese population identified some 12 loci as potential PCOS susceptibility gene candidates. One of these loci, the DENND1A locus at 9q22.32, has been replicated in GWAS studies in both Asian and European populations. Strauss and colleagues recently demonstrated DENND1A expression in testes, ovarian theca cells, and H295 adrenal carcinoma cells - all androgen producing tissues/cells. DENND1A is therefore an exceedingly strong PCOS susceptibility gene candidate. However, it remains unclear how DENND1A may contribute to the PCOS phenotype.

Importantly, there are two transcriptional forms of the DENND1A gene - a consequence of alternate splicing. The larger transcript (DENND1A Variant 1, or DENND1A.V1) encodes a 1009 amino acid protein, while the smaller transcript DENND1A.V2 encodes a truncated 559 amino acid. The product of DENND1A.V2 contains the DENN domain and a clathrin-binding domain, but differs from the product of DENND1A.V1 in two ways: (1) it does not contain the proline-rich C-terminal domain present in Variant 1, and (2) DENND1A.V2 results from differential splicing and contains a unique 33 amino acid C-terminal sequence. Of significant interest, published studies by Drs. McAllister and Strauss (McAllister JM, et al. Proc Natl Acad Sci USA. 2014;111:E1519-27) strongly implicate the DENND1A.V2 splice variant as a contributor to excessive theca cell androgen production in PCOS:

• Expression of DENND1A.V2 protein in cultured theca cells isolated from women with PCOS was over 3-fold elevated compared to normal ovarian theca cells. Similarly, DENND1A.V2 mRNA abundance was elevated in PCOS theca and correlated with increased theca cell androgen (dehydroepiandrosterone [DHEA]) production.
• Forced expression of DENND1A.V2 in normal theca cells increased expression of CYP17A1 and CYP11A1 (genes for key steroidogenic enzymes) along with androgen/androgen precursor (e.g., 17OHP4, DHEA, testosterone) and progesterone production.
• Knockdown of DENND1A.V2 in cultured PCOS theca cells reduced CYP17A1 and CYP11A1 expression in addition to 17OHP4 and DHEA production.
• Treatment of cultured PCOS theca cells with anti-DENND1A.V2 IgG antibodies reduced expression of CYP17A1 and CYP11A1 mRNA expression as well as DHEA and 17OHP4 synthesis.

These data provide strong support to the contention that the DENND1A.V2 splice variant is a major factor underlying the phenotype of cultured PCOS theca cells and, thus, the etiology of ovarian hyperandrogenemia in PCOS. By extension, the DENND1A.V2 splice variant may be a significant contributor to peripubertal hyperandrogenemia, and it may explain much of the variable hyperandrogenemia observed in obese peripubertal girls. The investigators propose that, in some peripubertal girls, increased expression of DENND1A.V2 in ovarian and/or adrenal cells promotes excess androgen secretion with the advent of puberty (when ovarian stimulation by LH increases) and/or in the presence of obesity (e.g., enhanced insulin secretion, augmenting LH-stimulated ovarian androgen synthesis and/or ACTH-stimulated adrenal androgen synthesis).

Of great interest, preliminary data from 5 normal women and 6 women with PCOS suggested that urinary exosomal DENND1A.V2 mRNA was approximately 3-fold elevated in women with PCOS compared to normally-cycling controls (McAllister JM, et al. Proc Natl Acad Sci USA. 2014;111:E1519-27). Exosomes are small vesicles (40-100 nm) that contain nucleic acids shed by cells into blood and urine, and they are known to be a stable source of RNA. Thus, urinary exosomal DENND1A.V2 quantity may serve as a marker of DENND1A.V2 activity in the ovary/adrenal.

In these proposed studies, the investigators (including collaborators Drs. McAllister and Strauss) will obtain urine for exosomal DENND1A.V2 mRNA quantitation in addition to blood samples to measure androgen levels, LH and insulin (the latter two previously-demonstrated to predict free testosterone). The investigators will test the hypothesis that the quantity of urinary exosomal DENND1A.V2 mRNA is a strong and independent predictor of androgen levels in peripubertal girls.

To the investigators' knowledge, no data exist that tie a PCOS susceptibility gene candidate to pubertal hyperandrogenemia. The investigators propose to evaluate a novel biomarker (urinary exosomal DENND1A.V2 mRNA) as a predictor of free testosterone in pubertal girls. Evaluation of this biomarker in adolescents is highly innovative.

The present proposal will build on Aim 3 of Project 1 (NIH P50 HD28934), in which the investigators carefully assess the determinants of hyperandrogenemia in girls with peripubertal obesity. Specifically, Aim 3 is an intensive protocol involving (a) overnight frequent blood sampling for LH and insulin along with (b) ovarian and adrenal stimulation protocols over the subsequent 2 days. Some of the subjects participating in this proposed pilot study will also participate in Aim 3. Correlation between urinary exosomal DENND1A.V2 mRNA and ovarian/adrenal stimulation protocols will allow a preliminary assessment of urinary exosomal DENND1A.V2 mRNA as a non-invasive measure of ovarian/adrenal steroidogenesis - a potentially important clinical tool.

Peripubertal girls with hyperandrogenemia are believed to be at high risk for developing adolescent/adult PCOS, but since the manifestations of PCOS can overlap with the findings of normal puberty, the diagnosis of PCOS may be unreliable until later adolescence. If urinary exosomal DENND1A.V2 mRNA is a significant predictor of hyperandrogenemia in this cohort, future studies will determine the utility of urinary exosomal DENND1A.V2 as a diagnostic test to predict the development of PCOS across puberty. This is an innovative and exceedingly exciting prospect that may have significant potential implications for at-risk girls.