Urinary DENND1A.V2 as a Predictor of Pubertal Hyperandrogenemia
Urinary DENND1A.V2 as a Predictor of Pubertal Hyperandrogenemia
Polycystic ovary syndrome (PCOS) is a common disorder marked by hyperandrogenism,
 oligo-/anovulation, and subfertility. The precise causes of PCOS are unclear, but the
 pathophysiology involves complex genetic and environmental influences. Importantly, not all
 girls with obesity have HA, and free testosterone (T) concentrations are highly variable in
 this group. Luteinizing hormone (LH) and insulin concentrations are significant but only
 partial predictors of free T in girls with obesity; significant unexplained variability in
 free T suggests that additional factors contribute to HA in this population. Abnormalities of
 ovarian and adrenal steroidogenesis are likely contributors in this regard, but such
 abnormalities are difficult to quantify. Recent Genome Wide Association Studies have
 identified DENND1A as a PCOS susceptibility gene candidate. Preliminary in vitro data
 strongly implicate a DENND1A splice variant called DENND1A Variant 2 (DENND1A.V2) as a
 contributor to excessive theca cell androgen production in PCOS. The investigators' primary
 goal with the proposed pilot study is to determine the relationship between urinary exosomal
 DENND1A.V2 mRNA and free T concentrations in peripubertal girls. The investigators
 hypothesize that urinary exosomal DENND1A.V2 mRNA quantity is a significant and independent
 predictor of peripubertal hyperandrogenemia. In this study, the investigators will carefully
 phenotype peripubertal girls with and without hyperandrogenemia (primarily in the form of
 hormonal, maturational, and anthropometric measurements) in addition to measuring urinary
 exosomal DENND1A.V2 mRNA. As a primary analysis, the investigators will examine the
 relationship between morning free testosterone and urinary exosomal DENND1A.V2, controlling
 for previously-described partial predictors of free testosterone (LH, insulin) in addition to
 potential confounders (BMI z-score, bone age). These studies will provide important
 information regarding the etiology of HA in peripubertal girls. Ultimately, these data may
 lead to a non-invasive test of ovarian/adrenal steroidogenic activity and support the
 development of a diagnostic test for PCOS in high-risk peripubertal girls (e.g., those with
 obesity).
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.
List of Inclusion Criteria

 • Peripubertal girls, Tanner breast stages 1-5

 List of Exclusion Criteria

 -  Age < 8 or > 17 y

 -  Men and boys are excluded

 -  Inability to obtain proper consent/assent

 -  Atypical obesity

 -  Underweight: Underweight is defined as a BMI-for-age percentile < 5

 -  Positive pregnancy test or lactation

 -  Assessment during the luteal phase as suggested by a serum progesterone ≥ 1.5 ng/ml

 -  Virilization or a total testosterone > 150 ng/dl

 -  Excessively elevated DHEA-S: This will be defined as a DHEA-S > 1.5 times the
 age-appropriate upper limit of normal

 -  Congenital adrenal hyperplasia (CAH)

 -  Cortisol deficiency/excess

 -  Inadequately-treated or unstable thyroid dysfunction

 -  Significant hyperprolactinemia: Since mild elevations may be seen in girls with
 hyperandrogenemia or PCOS, elevations up to 30 (i.e., 1.5 times the upper limit of
 normal) will be accepted in such girls

 -  Significant chronic medical history: This includes a significant history of cardiac or
 pulmonary dysfunction (e.g., known or suspected congestive heart failure; asthma
 requiring intermittent systemic corticosteroids; etc.); history of renal insufficiency
 or durable electrolyte abnormalities; or a history of substantial liver disease. A
 history of liver test abnormalities will be allowed in two circumstances: (1) mild
 bilirubin elevations will be accepted in the setting of known Gilbert's syndrome; (2)
 mild transaminase (ALT, AST) elevations may be seen in obese girls, so stable
 elevations < 1.5 times the upper limit of normal will be accepted in this group.

 -  Uncontrolled type 2 diabetes mellitus: This will be reflected by a hemoglobin A1c >
 7.0%. Subjects with impaired glucose tolerance or a diagnosis of type 2 diabetes that
 is well-controlled with lifestyle management alone will be allowed to participate.

 -  Type 1 diabetes mellitus: Since subjects with type 1 diabetes invariably require
 exogenous insulin, they will not be allowed to participate.