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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 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 Pit1 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.