ZHX2 Research Articles

An intronic genetic variant of ZHX2 predicts response to pegylated interferon α therapy in HBeAg-positive chronic hepatitis B patients

ZHX2 plays a crucial role in host immunity and modulates hepatitis B virus (HBV) replication. However, its correlation with immunomodulator-related treatment, i.g., pegylated interferon α(PegIFNα), for HBV remains uncertain. To explore the link between single nucleotide polymorphisms (SNPs) in ZHX2 and response to PegIFNα therapy for chronic hepatitis B (CHB) patients, we conducted a retrospective study in 945 hepatitis B e antigen (HBeAg)-positive CHB patients with at least 48 weeks of PegIFNα treatment and 24 weeks of follow-up from two phase-IV, multicenter trials (Cohort 1, n = 238; Cohort 2, n = 707). Thirty-eight tag SNPs were selected across the whole ZHX2 gene region. A polygenic score (PGS) was constructed by integrating multiple SNPs. The associations of ZHX2 SNPs and PGS with treatment response were assessed in both cohorts and their combination. Among the 38 tag SNPs, ZHX2_rs17289471 (T>C) was significantly associated with combined response (CR, i.e., HBeAg seroconversion and HBV DNA level <3.3log10IU/mL) at week 72 in both cohorts. The CR rate at week 72 increased steadily from rs17289471 TT to CT and CC genotype carriers in both Cohort 1 (P = 0.002) and Cohort 2 (P = 0.025) as well as Cohort 1 + 2 (P = 3.50 × 10−4). Moreover, a PGS integrating ZHX2_rs17289471 and five other previously identified SNPs was further significantly associated with CR rate at week 72 in Cohort 1 (P = 2.38 × 10−6), Cohort 2 (P = 1.04 × 10−7) and Cohort 1 + 2 (P = 9.37 × 10−13). Overall, ZHX2_rs17289471 and PGS have the potential to predict response to PegIFNα treatment of HBeAg-positive CHB patients.

P0046ZHX2 DOWNREGULATION DUE INSERTIONS AND DELETIONS IN THE HAS2-ZHX2 INTERGENIC REGION PREDISPOSE TO PODOCYTE DISEASE RELAPSE

Abstract Background and Aims ZHX2 transcriptional factor is normally found in podocyte membrane forming complexes with ZHX1 and APA or ZHX3 and Ephrin B1. Altered ZHX2 expression disrupts these interactions and is related with the worsening of different primary glomerular diseases such as focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD). A small percentage of Hodgkin lymphoma (HL) patients develop nephrotic syndrome (NS). Studies of Reed-Sternberg cell line L-1236 reveals a chromosomal rearrangement that leads to ZHX2 downregulation suggesting a possible role of ZHX2 in the development of NS in these patients. Human podocyte disease relapse is common after a common cold probably mediated by cytokines released by immune cells and the rhinovirus. Our aim is to study how the presence of insertions and deletions (InDels) between HAS2 and ZHX2 in patients with MCD, FSGS and HL could alter ZHX2 expression and the implication in podocyte disease relapse after a common cold and in HL. Method Genomic DNA between HAS2 and ZHX2 (Chr8:122624000-124001000 from UCSC hg19 GRCh37) was sequenced from 28 NS patients and 27 controls using Agilent Custom capture and high throughput Illumina sequencing. The CLC Genomics software was used to identify InDels (3-20 bp) present exclusively in patients. One of the identified InDels was replicated in human podocytes using CRISPR Cas9 and homology directed repair technology to study changes in ZHX2. A common cold cytokine cocktail containing IL-2, IL-4R, IL-6, IL-10, INF-γ, TNF-α and ICAM-1 was injected into control (BALB/c, n=5) and ZHX2 deficient mice (BALB/cJ, n=5) (Dose X); podocyte specific ZHX2 deficient (ZHX2 flox/flox cre+/+, n=3) and floxed control mice (ZHX2 flox/flox, n=3) (dose X/15). Buffalo Mna (B. Mna) rats were also injected with the cytokine cocktail (X/50) to study relapse in FSGS (n=7). Cell supernatant from HL Reed Sternberg cells (L-1236) (200µg of total protein) was injected into BALB/c (n=5) and BALB/cJ mice (n=5) and kidney function was assessed. Results Multiple InDels were found exclusively in the patient population, two of them, shared by two or more patients. The insertion at 122,533,694 was presented in patients with MCD, FSGS and HL with NS and also in L-1236 cells. This insertion was replicated in human podocytes using CRISPR/Cas9 (CRISPR B). Another insertion noted both in patients and controls was generated for comparison (CRISPR A). A reduced ZHX2 expression was detected in CRISPR B but not in CRISPR A single cell clones. The shared insertion at 122,787,088 was presented within the ZHX2 gene intron 1. BALB/cJ mice has lower ZHX2 expression in liver and podocytes due to an insertion in intron 1. To study whether this insertion could be related with relapse of MCD and FSGS following a common cold, a cytokine cocktail was injected into BALB/cJ and BALB/c mice. BALB/cJ mice developed acute albuminuria after cytokine treatment (65.3±24.3 μg per 18h), but not control BALB/c mice (10.8±1.5 μg per 18h), when compared with baseline values (BALB/cJ 5.1±1.1 μg per 18h; BALB/c 6.5±1.1 μg per 18h) (p&amp;lt;0.05). BALB/cJ mice had also higher nuclear expression of ZHX1. The cytokine cocktail also induced albuminuria in ZHX2 flox/flox/cre+/+ mice but not in control ZHX2 flox/flox, suggesting that ZHX2 deficiency in podocytes is responsible of kidney injury after cytokine injection. To study common cold related relapse in FSGS, B. Mna rats were injected with a rat cytokine cocktail, showing a significant increase in proteinuria (61.5±4.2 mg per 18h) compared with baseline (47±4.1 mg per 18h). Cell culture supernatant from L-1236 was injected into BALB/cJ and BALB/c mice to study the effect of secreted soluble mediators in NS. L-1236 supernatant (200 µg) had a nephrogenic effect in ZHX2 deficient mice but not in controls. Conclusion InDels between HAS2 and ZHX2 genes presented in patients with MCD, FSGS and HL, alter ZHX2 expression and are related to relapse following a common cold and in HL.

ZHX2 inhibits proliferation and promotes apoptosis of human lung cancer cells through targeting p38MAPK pathway.

To investigate the effect of ZHX2 on lung cancer cells proliferation and apoptosis. The mRNA and protein expression of ZHX2 were detected by qRT-PCR and western blot, respectively. The human lung cancer cells were divided into Control, NC, ZHX2, SB, and ZHX2 + Ani groups. The cell proliferation was detected by CCK-8 assay and the cell migration and invasion were detected by Transwell assay. Cell apoptosis was detected by flow cytometry. Apoptosis and p38MAPK signaling pathway related proteins were detected by western blot. The nude mice model of lung cancer xenograft was constructed. The tumor volume and tumor weight were measured. The expression of PCNA protein in tumor tissues was detected by immunohistochemistry. The apoptosis of tumor cells was detected by TUNEL staining. The ZHX2 and p38MAPK signaling pathway related proteins in tumor tissues were detected by western blot. The expression of ZHX2 gene and protein in the cancer cell lines were significantly decreased. Compared with control and NC groups, the cells proliferation, migration and invasion were inhibited in ZHX2 and SB groups, while the apoptosis and apoptosis related proteins were increased (p< 0.05). Meanwhile, compared with ZHX2 group, the tumor growth rate, volume, weight, the percentage of PCNA-positive cells, and p-P38 MAPK/P38 MAPK were increased significantly in ZHX2 + Ani group, while the apoptotic index and the expression of MMP-9 protein were significantly decreased (p< 0.05). ZHX2 could inhibit proliferation and promote apoptosis of lung cancer cells by inhibiting p38MAPK signaling pathway.

The role of Zhx2 transcription factor in bipolar cell differentiation during mouse retinal development

We found that the Zhx2 gene (whose product is known to act as a tumor suppressor in hepatocellular carcinoma) is expressed in embryonic retinal progenitors and in developing cone bipolar cells in the postnatal retina, as well as in Müller glia in the mature retina. To examine the functions of Zhx2 protein during retinal development, we performed loss- and gain-of-function analyses using a retinal explant culture system. We introduced a plasmid encoding Zhx2 shRNA into isolated mouse retinas at E17.5, and the retinas were cultured as explants. After 3 days of culture, proliferation was slightly enhanced, leading to retinas thicker than in the control, but this phenomenon was observed only transiently. After 14 days of the culture, the thickness and gross morphology of retinas expressing sh-Zhx2 were indistinguishable from those of the control. The numbers of rod cells, amacrine cells, and Müller glia were the same in both groups. However, although the total number of bipolar cells was the same, the experimental group saw an increased population of ON bipolar cells, and decreased numbers of a subset of OFF bipolar cells. We also examined the effects of overexpression of Zhx2. Although Zhx2 acts as a tumor suppressor, its overexpression in developing retinas did not lead to any discernible difference in retinal thickness, suggesting that proliferation activity was not affected. After 14 days of explant culture, the total number of bipolar cells decreased, and subset composition was altered. Taken together, these results suggest that Zhx2 plays roles in the regulation of bipolar cell subset fate determination during retinal development.

Zinc Fingers and Homeoboxes 2 (Zhx2) Regulates Sexually Dimorphic Cyp Gene Expression in the Adult Mouse Liver

The mammalian cytochrome P450 (Cyp) gene family encodes a large number of structurally related enzymes that catalyze a variety of metabolic and detoxification reactions. The liver is the primary site of Cyp expression in terms of expression levels and number of expressed genes, consistent with this organ's essential role in metabolism of endogenous and xenobiotic compounds. Many Cyp genes exhibit sexually dimorphic expression. For example, Cyp2a4 is expressed significantly higher in the adult liver of female mice compared to male mice. An exception to this pattern is seen in BALB/cJ mice, where male hepatic Cyp2a4 mRNA levels are substantially elevated compared to male mice of other strains. The Zinc fingers and homeoboxes 2 (Zhx2) protein governs the silencing of several genes in the postnatal liver, including α-fetoprotein, H19, and glypican 3. Zhx2 also regulates numerous hepatic genes that govern lipid homeostasis. We previously showed that the Zhx2 gene is mutated in BALB/cJ mice, which led us to consider whether elevated male hepatic Cyp2a4 levels in this strain are due to this Zhx2 mutation. Using mice with a conditional Zhx2 deletion, we show here that the absence of Zhx2 in hepatocytes results in increased Cyp2a4 expression in adult male liver. We extend this finding to show that additional Cyp genes are disregulated in the absence of Zhx2. We also show that mRNA levels of Cyp2a4 and several other female-biased Cyp genes are increased, and male-biased Cyp4a12 is decreased in mouse liver tumors. These data indicate that Zhx2 is a novel regulator of sex-biased Cyp gene expression in the normal and diseased liver.

ZHX2 and cancer

Transcription repressor ZHX2 is one of the members in ZHX protein family,which exists widely in human tissue and participates in the occurrence and development of various diseases.Researches show that ZHX2 is closely related to the occurrence and development of cancers,such as liver cancer,multiple myeloma,gastric cancer and colorectal cancer,which has the potential value of tumor treatment. Key words: Zinc fingers; Homeodomain proteins; Neoplasms; Transcription factors

Exome sequencing in familial corticobasal degeneration

BackgroundCorticobasal degeneration (CBD) is a neurodegenerative, sporadic disorder of unknown cause. Few familial cases have been described. ObjectiveWe aim to characterize the clinical, imaging, pathological and genetic features of two familial cases of CBD. MethodsWe describe two first cousins with CBD associated with atypical MRI findings. We performed exome sequencing in both subjects and in an unaffected first cousin of similar age. ResultsThe cases include a 79-year-old woman and a 72-year-old man of Native American and British origin. The onset of the neurological manifestations was 74 and 68 years respectively. Both patients presented with a combination of asymmetric parkinsonism, apraxia, myoclonic tremor, cortical sensory syndrome, and gait disturbance. The female subject developed left side fixed dystonia. The manifestations were unresponsive to high doses of levodopa in both cases. Extensive bilateral T1-W hyperintensities and T2-W hypointensities in basal ganglia and thalamus were observed in the female patient; whereas these findings were more subtle in the male subject. Postmortem examination of both patients was consistent with corticobasal degeneration; the female patient had additional findings consistent with mild Alzheimer's disease. No Lewy bodies were found in either case. Exome sequencing showed mutations leading to possible structural changes in MRS2 and ZHX2 genes, which appear to have the same upstream regulator miR-4277. ConclusionsCorticobasal degeneration can have a familial presentation; the role of MRS2 and ZHX2 gene products in CBD should be further investigated.

Lack of association of G779A ZHX-2 gene variant with HbF levels in β-thalassemia major

The inherited disorders of hemoglobin synthesis are the most common monogenic disorders worldwide. They include thalassemias, hemoglobin variants, and hereditary persistence of fetal hemoglobin. β-thalassemia is the most common monogenic disorder in India. Clinical manifestations of β-thalassemia are extremely variable in severity. The reasons for this heterogeneity are not very well understood. Previous studies have shown that the genetic variants that modulate HbF levels have a very strong impact on ameliorating the clinical phenotype. In the present study, 5570 blood samples from suspected cases were analyzed using HPLC, amplification refractory mutation system-PCR and reverse dot blot techniques. Of 5570 individuals, we found 676 cases of β-thalassemia disease. Molecular analysis revealed the presence of different β-thalassemia mutations in the population under study. Patients with β-thalassemia were classified into mild, moderate, and severe according to severity score based on Hb level, age of onset, age at which patients received their first blood transfusion, degree of growth retardation and splenectomy. Patients with β-thalassemia were analyzed for zinc finger and homeoboxes 2 (ZHX2) G779A polymorphism, and the association between ZHX2 gene polymorphism and severity of β-thalassemia was evaluated. We did not find a significant difference in genotypic and allelic frequency of ZHX2 gene between mild and moderate, mild and severe, and moderate and severe cases. There was no significant difference in high and low percentage of HbF in GG, GA, and AA bearing individuals showing that ZHX2 gene variant has no role in ameliorating the severity of β-thalassemia major in the South Indian population from Andhra Pradesh.

ZHX2, CHC1L and RAN Gene Expression Levels Determine Different Prognosis Groups in Multiple Myeloma (MM) Patients.

Gene Expression Profiling through RNA arrays has provided new clues to Multiple Myeloma pathogenesis and prognostic pattern evaluation. Recently, ZHX2, CHC1L & RAN expression have been highlighted as key elements in MM. In the present paper, we have evaluated theses genes by real time quantitative RT-PCR (RQ-PCR) in purified plasma cells from 74 patients with plasma cell discrasias.MATERIAL AND METHODS: Purified Bone Marrow cells were obtained from the following patients: 6 MGUS, 7 smoldering MM, 59 newly diagnosed symptomatic MM patients and 2 Plasma cell leukemia (PCL). After RNA extraction, RQ-PCR of CHC1L(C), RAN(R) and ZHX2 (Z) genes was carried out using the standard protocol from TaqMan® gene expression Assays-on-Demand in an ABI-PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA).Expression levels were normalized with ABL gene and expressed in n-fold times compared to the expression in a pool of RNA from mononuclear cells from healthy donors. The expression level of the different genes was evaluated for correlation with the diagnosis, clinical characteristics and prognosis of the patients.RESULT AND CONCLUSIONS: None of these genes displayed a clear relationship with the different stages of disease pathogenesis, although ZHX2 gene was slightly more expressed in the indolent forms of the proliferative disorders (MGUS and SMM). Within symptomatic MM patients, several interesting associations were observed. Thus, in hyperdiploid MM cases, CHC1L expressions observed were fewer than in those with a normal DNA index, confirming the participation of the gene product in chromosomal condensation during the mitosis. No other important associations were observed for this gene, although patients with the lowest expression displayed a very good prognosis, but without reaching statistically significant differences. As expected, RAN expression was related to S-Phase PC, since patients with high S phase values (>1.8 %) displayed higher levels of RAN transcripts. This, however, only resulted in a marginal impact on survival. ZHX2 provided the most interesting results, whereby decreased levels of ZHX2 were related to unfavorable prognostic indicators such as B2 microglobulin >4 mg/L and Hemoglobin levels <10.5 g/dl. This was significantly associated with a shorter survival. As such, patients with a level of ZHX2 expression lower than twice the expression of controls were associated with a very low survival (median of 6 months vs not reached, p=0.019). If we take into account the survival prediction value of these three genes, the following prognosis groups were defined: very good prognosis (Z+ C−), poor prognosis (Z− C+) and intermediate (the remaining patients).SUMMARY: From this study we can confirm that RAN, CHC1L and especially ZHX2 genes play a role in the pathogenesis and behavior of MM, this could be helpful in predicting survival of patients.

Hereditary persistence of alpha-fetoprotein and H19 expression in liver of BALB/cJ mice is due to a retrovirus insertion in the Zhx2 gene.

The alpha-fetoprotein (AFP) and H19 genes are transcribed at high levels in the mammalian fetal liver but are rapidly repressed postnatally. This repression in the liver is controlled, at least in part, by the Afr1 gene. Afr1 was defined >25 years ago when BALB/cJ mice were found to have 5- to 20-fold higher adult serum AFP levels compared with all other mouse strains; subsequent studies showed that this elevation was due to higher Afp expression in the liver. H19, which has become a model for genomic imprinting, was identified initially in a screen for Afr1-regulated genes. The BALB/cJ allele (Afr1(b)) is recessive to the wild-type allele (Afr1(a)), consistent with the idea that Afr1 functions as a repressor. By high-resolution mapping, we identified a gene that maps to the Afr1 interval on chromosome 15 and encodes a putative zinc fingers and homeoboxes (ZHX) protein. In BALB/cJ mice, this gene contains a murine endogenous retrovirus within its first intron and produces predominantly an aberrant transcript that no longer encodes a functional protein. Liver-specific overexpression of a Zhx2 transgene restores wild-type H19 repression on a BALB/cJ background, confirming that this gene is responsible for hereditary persistence of Afp and H19 in the livers of BALB/cJ mice. Thus we have identified a genetically defined transcription factor that is involved in developmental gene silencing in mammals. We present a model to explain the liver-specific phenotype in BALB/cJ mice, even though Afr1 is a ubiquitously expressed gene.

The mouse zinc-fingers and homeoboxes (ZHX) family; ZHX2 forms a heterodimer with ZHX3

Human zinc-fingers and homeobo xes (ZHX) 1, ZHX2 and ZHX3, members of the ZHX family, contain two Cys 2–His 2-type zinc-finger motifs and five homeodomains (HDs). These proteins not only form homodimers but heterodimers with ZHX1 as well and act as ubiquitous transcriptional repressors. The cloning of mouse ZHX2 and ZHX3 cDNAs and the corresponding genes from a 129 mouse genomic library are reported, along with an analysis of the heterodimerization of ZHX2 with ZHX3. The mouse ZHX2 and ZHX3 proteins consist of 836 and 951 amino acid residues, respectively. The similarity of amino acid sequences of each protein with those of human orthologue is 87.0% and 85.2%, respectively. An analysis of genomic clones revealed that an entire coding sequence and a portion of the 5′- and 3′-noncoding sequence of mouse ZHX2 cDNA are encoded by a single exon of the mouse ZHX2 gene as well as the mouse ZHX1 gene. In contrast, in the case of the mouse ZHX3 gene, the coding sequences of ZHX3 cDNA are separated by an intron. A 4.5-kb ZHX2 transcript, and three ZHX3 transcripts, 9.5-, 6.5- and 4.4-kb, are ubiquitously expressed, although their levels vary. Lastly, in vitro and in vivo protein–protein interaction assays revealed that ZHX2 is able to form a heterodimer with ZHX3 via a region containing each HD1.