Transcript Style B 24 by 48 wide - University of Chicago

Molecular Basis of Hormone Deficiency
J. Curley, E. Rochowicz-Wirthwein, J. Robbins, mentor - S. Radovick
University of Chicago Children’s Hospital
Introduction
Methods
Methods - cont.
Results - cont.
Conclusion
Prop-1 Gene
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 celltypes, and a mutation or deletion of the genes
encoding these factors has been shown to result in
anterior pituitary hormone deficiency in mammals.
Rpx Pitx
Rathke's
Pouch
-GSU
Lhx3/Lhx4
Prop-1
(Prop-1)
SF-1
GATA2
NeuroD1
corticotroph
Pit-1
GATA2
rostral
caudal
gonadotroph thyrotroph
ACTH
LH / FSH
lactotroph
PRL
TSH
FIGURE 1. Overview of anterior pituitary
somatotroph
GH
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.
Deficient
Factor
ACTH
def.
GH
def.
LH/FSH
def.
PRL
def.
TSH
def.
Rpx
Yes
Yes
Yes
Yes
Yes
Ptx-2
No
Yes
Yes
Yes
Yes
Lhx-3
No
Yes
Yes
Yes
Yes
Prop-1
No
Yes
+/-
Yes
Yes
Pit-1
No
Yes
No
Yes
Yes
The goal of this work was two-fold. First, to identify
novel mutations in these candidate genes encoding
pituitary-specific transcription factors in a pituitaryhormone deficient patient. Second, to determine the
mechanism by which any mutation causes
hypopituitarism.
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).
TABLE 2. Primers used in PCR of Prop-1.
Prop-1
Exon #1
Exon #2
Exon #3
Primer Sequence
sense 5’-GGAAGCAGAGAAATCTCAAGTC-3’
antisense 5’-AAAGCCAAGGGGTGCTCCAGTC-3’
sense 5’-TGGTCCAGCACCGAGGAGCGTC-3’
antisense 5’-TAATGCCCAACATTCTATGATAGC-3’
• 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 NaAc+50 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 @ 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.
Results
1
2
3
4
5
6
7
8
9 10
<-- a
<-- b
<-- c
sense 5’-GTGTCACCACCTATGTCAAGTGTG-3’
antisense 5’-TCCTAATCGGTGAGCTGACCCTCA-3’
• PCR products cleaned with Exo-Sap-IT to eliminate
primers, primer-dimers, and nucleotides before sending
for sequencing: 5 uL Shrimp Alkaline Phosphatase (SAP)
Buffer; 0.5 uL SAP; 0.5 uL E. coli Exonuclease I;37° x
45”,95° x 15”, store at 4°
• Exons directly or indirectly sequenced (next column)
at a DNA sequencing facility.
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 HILO 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.
A -> T (nt 343)
C G
A
T
T
T
T G
A A
A C C A A
A
T C
2143
Fragment
07F_Roch
_KYJO6_2
5’
3’
R50D
Del 112-114
0
C G
A
T
T
T
T G
A A
A C C A A
A
T C
81 82
83
84
85 86
87 88
89 90
91 92 93 94 95
96
97 98
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.
Rpx1 exon 4
Ptx2 exon 2
Ptx2 exon 6
Ptx2 exon 7
Prop-1 exon 1
Prop-1 exon 2
Prop-1 exon 3
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype (C336T)
Wildtype
Mutation A142T
R73C
R73H
S109X
F117I
P164X
S83P
F88S
R99X
R99Q
P160X
R120C
FIGURE 5.
Known mutations in the paired-like
homeodomain transcription factor Prop-1 (prophet of Pit1).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.
Support/IRB information
RO1 DK 53977 Differential Regulation of Pit-1 Responsive Genes by CBP
K24 DK 01362 Hypopituitarism: clinical and molecular characterization
Figure 4. WT Prop-1 and
1
2
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.
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.
IRB Approval 2/5/02; IRB Protocol # 10838B
References
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bVallette-Kasie,
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cGuy,
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L.E., Radovick, S. Other transcription factors and hypopituitarism.
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