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1
Adolescent Polycystic Ovary Syndrome (PCOS) is a
Precursor of Adult PCOS and Glucose
Intolerance B.D. Bordini, E.E. Baumann, and R.L
RosenfieldSection of Pediatric Endocrinology
Abstract
Results
Summary

• PCOS and Endocrinologic Heterogeneity
• Hyperandrogenemia in PCOS arises from excess
  production of male hormone from the ovaries
  (functional ovarian hyper-androgenism, FOH),
  adrenal gland (functional adrenal
  hyperandrogenism, FAH), or both.
• Hyperinsulinemic insulin resistance aggravates
  the androgen excess of PCOS.

• By 29 years of age, 9/22 recalled adolescent
  patients with PCOS demonstrated the following
  characteristics
• 7/9 remained clearly hyperandrogenic
• 6/9 had abnormal glucose tolerance (representing
  27 of the original adolescent cohort and 37.5
  of the FOH cohort)
• One had T2DM
• One patient had developed endometrial carcinoma

PCOS is a poorly understood syndrome of chronic
hyperandrogenism and anovulation in which
hyperinsulinemia, related to type 2 diabetes
mellitus (T2DM) plays an important pathogenic
role. Adolescents with PCOS are more insulin
resistant (IR) than age- and weight-matched
controls. Since normal adolescence is a stage of
transient IR, we have tested the hypothesis that
improvement of IR with age may ameliorate the
glucose intolerance and androgen excess of
adolescent PCOS. A cohort of 23 hyperandrogenic
adolescents were previously studied in 1989-1994
at 10-17 years of age, and were reported as
having clinical and endocrine features similar to
contemporaneously studied adults with classical
and nonclassical PCOS. Of these, 16 had the
typical PCOS type of functional ovarian
hyperandrogenism (FOH) and the remainder had
either idiopathic or functional adrenal
hyperandrogenism (FAH). Of the FOH subgroup, 12
had obesity, 12 had acanthosis nigricans, and 3
had impaired glucose tolerance (IGT), all
suggestive of hyperinsulinemia. Recall letters
were sent to all. 4 with FOH and 1 with FAH
could not be located (of the remaining 17, 9
were restudied at 18 to 29 years of age 8 were
in the FOH subgroup and 1 had FAH). All
underwent androgen sampling and an oral glucose
tolerance test (OGTT). From the OGTT data, we
derived an insulin-resistance index (IRI) by the
homeostatic model assessment method fasting
insulin (µIU/ml) x fasting plasma glucose
(mmol/L) / 22.5. BMI increased from an average
of 33.2 to 40.3 kg/m2 (plt0.05). 7/9 remained
oligo-anovulatory. 7/9 were hyperandrogenic, one
had a top-normal plasma free testosterone and may
have been on estrogen at the time and the only pt
with FAH had a normal free testosterone level.
Glucose metabolism deteriorated, 6 of 9 now had
an abnormal OGTT (5 IGT, 1T2DM). Average IRI did
not change (mean IRI9.1). One who had not
consistently taken cyclic progestin developed
endometrial carcinoma at 22 years of age. We
conclude that by 29 years of age, 7/9 recalled
adolescents remained hyperandrogenic. 6/8
recalled FOH patients now had abnormal glucose
tolerance. This represents almost 40 of the
original FOH cohort. Even if none of the
nonresponding patients had abnormal glucose
tolerance, at least on quarter (6/23) of the
original cohort of adolescent PCOS patients had
developed abnormal glucose tolerance, with one
T2DM, by early adulthood. One patient already
had endometrial carcinoma. Patients with
adolescent PCOS are thus at high risk for serious
adult disease, including T2DM.
Mean IRI for age matched norm group was 6.24
(0-13.62) Mean normal free testosterone level was
6.5 (0.9-12) Recall Population in Relation to
PCOS Sub-type FOH /- FAH (n17) 8
restudied 4 lost (no forwarding address)
5 no response FAH only (n6) 1 restudied
1 lost (no forwarding address) 4 no
response Respondents thus consisted of re-called
study group of 8 adults with FOH and 1 with
FAH 7/8 patients with FOH were hyperandrogenic, 1
had top-normal plasma free testosterone
(10pg/ml) The patient with FAH had normal free
testosterone level Average insulin resistance
index did not change significantly Glucose
metabolism deteriorated 6/9 had an abnormal
OGTT BMI increased in 8/9 patients with an
average rise from 33.3 to 40.3 kg/m2 (plt0.05) 7/9
remained oligo-anovulatory (history not available
from other 2) One patient who had inconsistently
taken cyclic progestin developed endometrial
carcinoma at age 22
Hypothesis
Improvement of insulin resistance with age may
ameliorate the hyperandrogenemia and glucose
intolerance of patents diagnosed with PCOS as
adolescents.
Specific Aim

• Reevaluate adolescents diagnosed with PCOS from
  1989 to 1994.
• Examine for PCOS and DM

Conclusion

• Adolescent PCOS persists into early adulthood
• Normalization of elevated androgens or abnormal
  glucose metabolism in adolescents with PCOS
  seldom if ever occurs after sexual maturation
• Adolescent PCOS confers a high risk for adult
  metabolic syndrome, including T2DM

Methods
Background

• Study protocol of adolescents with PCOS symptoms
  (1989-1994)
• Twenty-two consecutive hyperandrogenic girls
  diagnosed in Pediatric Endocrine Clinic
• 9.9 - 17.5 years old
• Source localization using adult criteria
• -FOH GnRH agonist test or
  dexamethasone test critieria
• -FAH ACTH test criteria
• Oral glucose tolerance test (OGTT)
• Follow up protocol
• Recalled at 18-29 years of age
• - 3.35 - 13 years follow up
• - All 6-16 years post-menarche
• Interim history was obtained
• Plasma androgen battery (free testosterone)
• Oral glucose tolerance test (OGTT)
• Homeostatic model assessment (HOMA), from which
  we derived an insulin-resistance index
  IRIfasting plasma insulin (µIU/ml) x fasting
  plasma glucose (mmol/L) / 22.5
• Age-matched norms protocol
• All girls recruited from the local neighborhood
  population
• Aged 10.3 to 16.7 years
• Screened to ensure they had no symptoms of PCOS

Preliminary Data for Recalled Patients

• PCOS and Hyperandrogenemia
• PCOS is a heterogeneous disorder, characterized
  by the pubertal onset of hyperandrogenemia and
  chronic anovulation.
• Apter, et al. noted that androgen levels in
  healthy teenagers correlate with adult levels and
  are inversely associated with fertility
• Does androgen excess of adolescent PCOS persist?
• PCOS, insulin resistance and type 2 diabetes
  mellitus (T2DM)
• Hyperinsulinemic insulin resistance is a
  significant factor in PCOS
• -Insulin resistance and T2DM are 5-7 times more
  prevalent in PCOS than in the general population.
• The insulin resistance of normal adolescence
  improves with sexual maturation.
• Do the insulin resistance and glucose intolerance
  of adolescent PCOS improve or worsen, becoming
  typical of adult PCOS?

Future Evaluations

• After obtaining IRB approval, we will expand the
  number of recall patients
• We will compare the entire recall groups lab
  findings to a group of age-matched adult norms

2
Molecular Basis of Hormone Deficiency J. Curley,
E. Rochowicz-Wirthwein, J. Robbins, mentor - S.
Radovick University of Chicago Childrens Hospital
Introduction
Methods
Methods - cont.
Results - cont.
Conclusion
Pituitary development and hormone expression
in mammals is controlled by pituitary-specific
transcription factors including Hesx-1 (Rpx),
Ptx-2, Lhx-3, Prop-1, and Pit-1. These factors
initiate a cascade of development events
resulting in mature pituitary cell-types, and a
mutation or deletion of the genes encoding these
factors has been shown to result in anterior
pituitary hormone deficiency in
mammals. FIGURE 1. Overview
of anterior pituitary development.d Mutations
in these genes encoding pituitary-specific
transcription factors contribute to the growth
hormone deficient phenotype (i.e. short stature)
/- other pituitary hormone deficiency phenotypes
by interrupting the cascade of development and
maturation of the pituitary-cell types and thus,
cause hormone deficiency(ies) (GH /- ACTH,
LH/FSH, TSH, and PRL) in these patients.
TABLE 1. Hormone deficiencies associated with
the transcription factors investigated in this
work. The goal of this work was
two-fold. First, to identify novel mutations in
these candidate genes encoding pituitary-specific
transcription factors in a pituitary-hormone
deficient patient. Second, to determine the
mechanism by which any mutation causes
hypopituitarism.

• Direct Sequencing 5 uL PCR product, 1 uL
  primer (sense OR antisense), and 4 uL ABI prism
  sequencing solution.Then, sequence reaction on
  PCR machine. Finally, purify
• 2 uL 3 M NaAc50 uL 95 EtOH-vortex, ice x10,
  cold centrifuge x 20, decant supernatant, add
  250 uL 70 EtOH cold centrifuge x 5, decant
  supernatant, dry pellet
• Indirect sequencing
• Ligation Ligate PCR product into pTOPO with
  ECOR1 on each side 1 uL TOPO vector, 1 uL PCR
  product, 1 uL salt solution, and 1 uL H2O
  incubate 30 min at room temperature
• Transformation 100 uL thawed DH5-? cells 3
  uL Ligation mix, then ice x 15 to shock,
  incubate _at_ 37 x 45 sec then return to ice x 2
  900 uL LB shaker X 1 100 ul IPTG xGAL
  culture plates (w/ Amp)- 37 overnight 3 mL
  Circle Grow(Amp) white colony Shaker
  overnight
• Miniprep yields DNA then mix 4 uL with 4 uL H2O,
  1 uL buffer H, 1 uL ECOR1and leave 1 hour at 37
• Sequencing reaction with T7 solution
• Compare Sequences to published exon sequences
  (NCBI Sequence viewer-website) and wild type
  sequences.

• I. PATIENT XX, PHENOTYPE
• Blood obtained from patient who was an ex 34 week
  infant of nonconsanguinous parents. In his
  neonatal period he had hypoglycemia, seizures,
  micropenis, and prolonged indirect
  hyperbilirubinemia. He had the following
  evaluation
• cortisol 1.54 mcg/dl
• on ACTH stim test, basal cortisol was 3.6 mcg/dl
  and at 6 hrs was 3.44 mcg/dl.
• LH/FSH - no information
• TSH 0.07 mIU/ml (0.35-5.5)
• PRL 36 ng/dl (12-27)
• OPTIC NERVE - no information
• XX was started on hydrocortisone, Na thyroxine,
  and synthetic growth hormone therapy.
• II. CANDIDATE GENES PROBED
• Genomic DNA made w/Qiagen Flexigene DNA Kit
• PCR conditions optimized for each exon
• i.e. buffers, MgCl2
• Invitrogen Optimizer Setting with varied
  annealing temperatures
• Individual or consecutive exons of candidate
  genes amplified by PCR (Table 2).

R50D
Del 112-114
FIGURE 5. Known mutations in the paired-like
homeodomain transcription factor Prop-1 (prophet
of Pit-1).d A142T lies just outside of the
homeodomain (black shading) of Prop-1 in exon 3.
This would be near a conserved basic region, B2,
required for nuclear localization, DNA binding,
and target gene activation. Other possible
effects could include transcription modification
via changed tertiary structure. A142T is
noted to be a possible polymorphism. Given
geographical association of this reported
polymorphism,a the substitution could be a
mutation with a discernable change in function.
Accordingly, transfection assays are underway to
combine with a luciferase reporter system and
probe function quantitatively, and initial
results are promising. Parental samples have
been obtained for analysis. Limitations of this
work include incomplete patient description as
well as other candidate genes which are not yet
assayed.
Rpx
Pitx
a-GSU
Lhx3/Lhx4

FIGURE 3. Rpx 1 exon 4 is wild type in patient
XX. The PCR products (i.e. from gel such as in
Figure 2) were sequenced and compared to the
published exon sequences. Shown here is an
assembling and confirmation that XX has the wild
type sequence for part of RPX1 exon 4 (confirmed
throughout sequence). Table 3. Of the sequences
optimized and analyzed, a possible mutation in
Prop-1 was found.
DNA-binding tested in gel shift assays utilizing
radiolabeled consensus DNA-binding elements and
protein translated in reticulocyte lysate to
assess the ability of wild type and mutant
proteins to bind to known response elements. A
typical TNT T7 kit recipe follows 40 ?L lysate,
1.5 mcg DNA, 1 ?L met, 7.5 ?L water, mix, spin,
30o x 1-2 hrs, spin. Probe was 15 ?L DIDC, 1.5
?L 0.1 M DTT, 2 ?L PRDQ9 probe P-32, 130 ?L BSB
buffer. Lanes 1 and 2 were loaded with 4 ?L
lysate mixture and 10 ?L probe.
Prop-1
Pit-1
(Prop-1) SF-1 GATA2
NeuroD1
GATA2
rostral
caudal
lactotroph
gonadotroph
thyrotroph
somatotroph
corticotroph
Results
1 2 3 4 5 6 7
8 9 10
Support/IRB information
lt-- a lt-- b lt-- c
FIGURE 2. Optimizing PCR conditions to
obtain introns of two transcription factors, Rpx1
and Ptx-2 success at 700 Bp for Ptx-2 exon 7
(band b) in this gel. Band a is DNA in the well,
and band c is Rpx primer-dimer. Lanes 1-2 are
HI-LO standard (Bionexus) lanes 3-6 are Rpx exon
3 sense and exon 4 antisense primers at 60o,
buffer M lanes 7-10 are Ptx-2 exon 7 sense and
antisense, standard buffer, 60o. PCR
reaction sample preparation 40 ?L H2O, 1 ?L
primer (sense), 1 ?L primer (anti-sense), 5 ?L
buffer, 4 ?L d NTP, 1 ?L DNA, and 0.5 ?L TAQ
Polymerase.
RO1 DK 53977 Differential Regulation of Pit-1
Responsive Genes by CBP K24 DK 01362
Hypopituitarism clinical and molecular
characterization IRB Approval 2/5/02 IRB
Protocol 10838B
1 2
Figure 4. WT Prop-1 and A142T bind target DNA to
approximately the same extent. In lane 1 WT
Prop-1, while lane 2 has the potential
mutation, A142T. Control lane with empty vector
is not shown but revealed no shift.
References
aNakamura, Y., Usui, T., Mizuta, H., Murabe, H.,
Muro, S., Suda, M., Tanaka, K., Tanaka, I.,
Shimatsu, A, and Nakao, K. Characterization of
Prophet of Pit-1 gene expressioin in normal
pituitary and pituitary adenomas in humans. J.
Clin. Endocrinology Met. 1999 84(6)
1414. bVallette-Kasie, S., Barlier, A.,
Teinturier, C., Diaz, A., Manavela, M.,
Berthezene, F., Bouchard, P., Chaussain, J. L.,
Brauner, R., Pellegrini-Bouiller, I., Jaquet, P.,
Enjalbert, A., Brue, T. Prop1 Gene screening in
patients with multiple pituitary hormone
deficiency reveals two sites of hypermutability
and a high incidence of corticotroph deficiency.
JCEM 2001 86(9) 4529. cGuy, J. C., Hunter, C.
S., Showalter, A.D., Smith, T. P. L.,
Charoonpatrapong, K., Sloop, K.W., Bidwell, J.
P., Rhodes, S. J. Conserved amino acid sequences
confer nuclear localization upon the prophet of
Pit-1 pituitary transcription factor protein
Gene 2004 336, 263. dCohen, L.E., Radovick, S.
Other transcription factors and hypopituitarism.
Reviews in Endo. Metobolic Disorders, 2002 3
301-311.