ZNF764 haploinsufficiency may explain partial glucocorticoid, androgen, and thyroid hormone resistance associated with 16p11.2 microdeletion

Abstract

Context: Nuclear hormone receptors exert their transcriptional effects through shared cofactor molecules; thus, defects in such intermediate proteins may be associated with multiple hormone resistance. Microdeletion of small chromosomal segments results in hereditary or sporadic diseases by affecting expression of residing genes.

Objectives: We describe a 7-yr-old boy with partial resistance to glucocorticoids, thyroid hormones, and possibly androgens. He was diagnosed as being in the autism spectrum disorder and had developmental delay and several facial morphological manifestations. We explored genes responsible for multiple hormone resistance of this case.

Results: We found in this patient an approximately 1.1-Mb heterozygous 16p11.2 microdeletion, which included an approximately 500-kb unique deletion along with the common, previously reported approximately 600-kb 16p11.2 microdeletion. The small interfering RNA-based screening revealed that knockdown of ZNF764, which is located in the deleted segment unique to our case, significantly reduced glucocorticoid-, androgen-, and thyroid hormone-induced transcriptional activity of their responsive genes in HeLa cells, whereas its overexpression enhanced their transcriptional activity. The activities of the estrogen and progesterone receptors, cAMP response element-binding protein, and p53 were not affected in these cells. ZNF764 (zinc finger protein 764) expression was reduced in the patient's peripheral blood mononuclear cells, whereas exogenously supplemented ZNF764 recovered responsiveness to glucocorticoids in the patient's Epstein-Barr virus-transformed lymphocytes. The effect of ZNF764 on the glucocorticoid receptor transcriptional activity was mediated through cooperation with a general nuclear hormone receptor coactivator, transcriptional intermediary factor 1.

Conclusions: ZNF764 haploinsufficiency caused by microdeletion may be responsible for the partial multiple hormone resistance observed in our patient. ZNF764 appears to be involved in glucocorticoid, androgen, and thyroid hormone action.

Fig. 1.

Clinical manifestations and an approximately 1.1-Mb heterozygous microdeletion at 16p11.2 of our patient. A–C, The patient has short stature and facial and genital abnormalities. Images of the patient (A) and his genital region showing a micropenis (B) and bifid scrotum (surgically repaired at 2 yr of age) (C) are shown. D, Ratio plot of patient's chromosome 16. The aCGH analysis using 1340 BAC clone probes identified a sharp ratio drop at the area of chromosome 16p11.2. E, Heterozygous loss of CTD-3159M2 in the FISH analysis. FISH was performed for the patient's chromosomes by using as a probe the CTD-3159M2 marker located in the deleted area. The affected chromosome 16 harboring the microdeletion and the intact chromosome 16 are indicated with arrowhead and arrow, respectively. F, The patient's 16p11.2 microdeletion spans approximately 1.1 Mb and contains 55 genes. Chromosome 16 (top), the area around the deleted segment found in the patient, and the genes harbored in it as well as results of quantitative PCR for representative genes are shown. The bottom red bar indicates the segment of deletion found in the patient. Bars indicate mean ± se values of relative dosage of indicated genes obtained by dividing levels of patient's copy number with that of normal controls. Dashed lines indicate dosages of 1.0 (presence of two alleles) and 0.5 (presence of only one allele), respectively. Chromosomal positions of some genetic markers used in the aCGH and FISH analysis are also shown.

Fig. 2.

mRNA levels of some glucocorticoid-, androgen-, and thyroid hormone-responsive genes and their responsiveness to respective hormones are reduced in patient's PBMCs. A–C, mRNA levels of some glucocorticoid-, androgen-, and thyroid hormone-responsive genes are reduced in patient's PBMCs. mRNA levels of known glucocorticoid-responsive genes (annexin A1, DUSP1, GILZ, PER1, and tristetraprolin) (A), androgen-responsive genes (FKBP5, IκBα, and SGK1) (B), thyroid hormone-responsive genes (KLF9, G6PD, and ME1) (C), and control RPLP0 were determined by SYBR Green-based real-time PCR. Bars represent mean ± se values of mRNA levels of the indicated genes corrected for those of RPLP0 by employing the levels of normal subjects as 1. *, P < 0.05; **, P < 0.01; n.s., not significant, compared with control. D, mRNA expression of the glucocorticoid-responsive (GILZ), androgen-responsive (SGK), and thyroid hormone-responsive (KLF9) genes demonstrated blunted response to their corresponding hormones in the patient's PBMCs. PBMCs obtained from the patient or three age- and sex-matched normal subjects were incubated in the presence or absence of 10⁻⁶ m dexamethasone (Dex), 10⁻⁸ m dehydrotestosterone (DHT), or 10⁻⁷ m T₃, and mRNA expression of GILZ, SGK, KLF9, and control RPLP0 were determined by SYBR Green-based real-time PCR. mRNA levels of respective genes were corrected for those of RPLP0, and fold expression was calculated by employing the values obtained in the absence of ligand as 1-fold, respectively, for the patient and control subjects. Bars represent mean ± se values of fold mRNA levels. **, P < 0.01, compared with control (the value of the control subjects obtained in the presence of indicated hormones). DUSP1, Dual-specificity phosphatase 1; FKBP5, FK506-binding protein 5; G6PD, glucose-6-phosphate dehydrogenase; IκBα, inhibitor of κBα; KLF9, Kruppel-like factor 9; ME1, malic enzyme 1; PER1, period 1; SGK1, serum/glucocorticoid regulated kinase 1.

Fig. 3.

Protein and mRNA levels of GR, AR, TRα, and TRβ are not altered in the patient. Protein (A) and mRNA (B) levels of GR, AR, TRα, and TRβ were determined with Western blots using specific antibodies or SYBR Green-based real-time PCR using total RNA extracted from PBMCs of the patient and six age- and sex-matched normal subjects. Bars represent mean ± se values of protein (A) and mRNA (B) levels of GR, AR, TRα, and TRβ corrected, respectively, for those of β-actin (A) and RPLP0 mRNA (B) by setting the levels of normal subjects as 1. *, P < 0.05; **, P < 0.01; n.s., not significant, compared with control. Representative gel images of the Western blot for GR, AR, TRα, and TRβ (top panels) and β-actin (bottom panels) are shown in the lower panels of A.

Fig. 4.

ZNF764 knockdown reduces transcriptional activity of GR, AR, and TRβ. A, ZNF764 knockdown reduces dexamethasone-stimulated GILZ mRNA expression in HeLa cells. HeLa cells were transfected with ZNF764 or control siRNA, treated with 10⁻⁶ m dexamethasone (Dex) and mRNA levels of glucocorticoid-responsive GILZ, ZNF764, and control RPLP0 was measured with SYBR Green-based real-time PCR. Bars represent mean ± se values of GILZ (left panel) or ZNF764 (right panel) mRNA levels corrected for those of the control RPLP0 mRNA. **, P < 0.01, compared with the control values with the same treatment. B and C, ZNF764 knockdown reduces ligand-stimulated transcriptional activity of AR and TRβ in HeLa cells. HeLa cells were transfected with ZNF764 or control cyclophilin B siRNA together with AR-expressing plasmid (B) or TRβ1-expressing plasmid (C) and MMTV-Luc (B) or DR4-Luc (C) in the presence of pGL4.73[hRluc/SV40]. Bars represent mean ± se values of the firefly luciferase activity normalized for renilla luciferase activity in the presence or absence of 10⁻⁸ m dehydrotestosterone (DHT) (B) or 10⁻⁷ m T₃ (C) in the left panels, whereas those of ZNF764 mRNA levels corrected for those of the control RPLP0 mRNA measured in the aliquots of samples are shown in the right panels. **, P < 0.01, compared with the control values with the same treatment.

Fig. 5.

ZNF764 expression is reduced in the patient's PBMCs and exogenous supplementation of ZNF764 normalizes responsiveness of the patient's EBV-transformed lymphocytes to dexamethasone. A and B, Protein and mRNA levels of ZNF764 are approximately 50% lower in patient's PBMCs than in those of normal controls. ZNF764 protein (A) and mRNA (B) levels were determined, respectively, with Western blots using anti-ZNF764 antibody and with SYBR Green-based real-time PCR using total RNA extracted from PBMCs of the patient and six age- and sex-matched normal subjects. Bars represent mean ± se values of ZNF764 protein (A) and mRNA (B) levels corrected, respectively, for those of β-actin (A) and RPLP0 mRNA (B) by setting the levels of normal subjects as 1. *, P < 0.05; **, P < 0.01; n.s., not significant, compared with control. Representative gel images of the Western blot for ZNF764 (top panel) and β-actin (bottom panel) are shown in the lower panels of A. C–E, Exogenous supplementation of ZNF764 recovers GR-induced transcriptional activity in patient's EBV-transformed lymphocytes. EBV-transformed lymphocytes of the patient or three normal subjects were transfected with control or ZNF764-expressing plasmid and were treated with 10⁻⁶ m dexamethasone (Dex). Bars represent mean ± se values of GILZ mRNA levels corrected for those of the control RPLP0 mRNA. **, P < 0.01; n.s., not significant, compared with the control value obtained in the presence of dexamethasone.

Fig. 6.

ZNF764 acts as a coactivator for GR, AR, MR, and TR by cooperating with TIF1β. A and B, ZNF764 overexpression enhances GR-induced transcriptional activity. HCT116 cells were transfected with increasing amounts (A) or 0.05 μg/well (B) of ZNF764-expressing plasmid together with MMTV-Luc and pGL4.73[hRluc/SV40]. Bars and circles represent mean ± se values of the firefly luciferase activity normalized for renilla luciferase activity in the presence or absence of 10⁻⁶ m dexamethasone (Dex) (A) or indicated concentrations of this steroid (B). C, ZNF764 enhances GR-, AR-, TRα-, TRβ-, and mineralocorticoid receptor (MR)-induced transcriptional activity but not of progesterone receptor (PR)-A, estrogen receptor-α (ERα), CREB, and p53. HCT116 cells were transfected with indicated steroid hormone receptor- or CREB-expressing plasmid together with their responsive promoter-driven luciferase gene and pGL4.73[hRluc/SV40] and were treated with indicated compounds or transfected with the plasmid expressing the α-catalytic subunit of the protein kinase A (PKA) or p53. Bars represent mean ± se values of the firefly luciferase activity normalized for renilla luciferase activity in the presence or absence of indicated compounds, PKA, or p53. **, P < 0.01; n.s., not significant, compared with the control value obtained in the presence of indicated steroids, PKA, or p53. D, Coexpression of TIF1β increased ZNF764-induced enhancement of GR transcriptional activity in HCT116 cells. HCT116 cells were transfected with increasing amounts of ZNF764-expressing plasmid together with MMTV-Luc and pGL4.73[hRluc/SV40] in the presence or absence of 0.01 μg/well of TIF1β-expressing plasmid. Bars represent mean ± se values of the firefly luciferase activity normalized for renilla luciferase activity in the presence or absence of 10⁻⁶ m dexamethasone (Dex). E, ZNF764 knockdown attenuated TIF1β-induced enhancement of GR transcriptional activity in HCT116 cells. HCT116 cells were transfected with the control cyclophilin B or ZNF764 siRNA together with MMTV-Luc and pGL4.73[hRluc/SV40] in the presence or absence of 0.1 μg/well of TIF1β-expressing plasmid. Bars represent mean ± se values of the firefly luciferase activity normalized for renilla luciferase activity in the presence or absence of 10⁻⁶ m dexamethasone (Dex).