Dr. Liu had extensive trainings in human molecular genetics, tumor biology, and experimental therapeutics. She published more than 20 peer-reviewed manuscripts, including Nature Genetics, Blood, Leukemia, and Journal of Clinical Investigation (JCI). She was a co-investigator in several NIH grants in her previous Institutes. In 2019, Dr. Liu joined the Department of Pediatrics as a Senior Research Scientist. Dr. Liu is one of the world-known experts in research on a rare children leukemia, juvenile myelomonocytic leukemia (JMML). Her current research is focusing on the dysregulated developmental hematopoiesis in leukemia transformation and development of mouse models for Diamond-blackfan anemia (DBA).
Current Role at Stanford
Senior Research Scientist
Education & Certifications
PhD, Peking Union Medical College (PUMC) Beijing, China, Medical Oncology (Clinical Pharmacology) (1991)
MD, West China School of Medicine, Chengdu, China, Medicine (1983)
JMML, Developmental hematopoiesis, Leukemia Transformation in MDS/MPN, Diamond-blackfan anemia, and Ribosomopathies
Professional Affiliations and Activities
Executive Committee Member, Chinese American Biopharmaceutical Society (CABS) (2019 - Present)
Life Member, Chinese American Hematologist and Oncologist Network (CAHON) (2007 - Present)
Active Member, Society for Clinical and Translational Science (2009 - Present)
Active Member, American Society of Hematology (2003 - Present)
Animal models of Diamond-Blackfan anemia: updates and challenges.
Diamond-Blackfan anemia (DBA) is a ribosomopathy that is characterized by macrocytic anemia, congenital malformations, and early onset during childhood. Genetic studies have demonstrated that most patients carry mutations in one of the 20 related genes, most of which encode ribosomal proteins (RP). Treatment of DBA includes corticosteroid therapy, chronic red blood cell transfusion, and other forms of immunosuppression. Currently, hematopoietic stem cell transplantation is the only cure for DBA. Interestingly, spontaneous remissions occur in 10-20% of transfusion-dependent DBA patients. However, there is no consistent association between specific mutations and clinical manifestations. In the past decades, researchers have made significant progress in understanding the pathogenesis of DBA, but it remains unclear how the ubiquitous RP haploinsufficiency causes the erythroid-specific defect in hematopoiesis in DBA patients, and why there is a difference in penetrance and spontaneous remission among individuals who carry identical mutations. In this paper, we provide a comprehensive review of the development of DBA animal models and discuss the future research directions for these important experimental systems.
View details for DOI 10.3324/haematol.2022.282042
View details for PubMedID 36384250
Sustained fetal hematopoiesis causes juvenile death from leukemia: evidence from a dual-age-specific mouse model.
2020; 4 (15): 3728–40
It is not clear whether disrupted age-specific hematopoiesis contributes to the complex manifestations in leukemia patients who carry identical mutations, particularly in pediatric and adult patients with similar clinical characteristics. By studying a dual-age-specific mouse model, we demonstrate that (1) loss of Pten during the fetal-to-adult hematopoiesis switch (hematopoiesis switch) causes sustained fetal hematopoiesis, resulting in death in juvenile leukemia; (2) myeloid-biased hematopoiesis in juvenile mice is associated with the sustained fetal properties of hematopoietic stem cells (HSCs); (3) the age specificity of juvenile myelomonocytic leukemia depends on the copy number of Pten and Nf1; (4) single-allelic Pten deletion during the hematopoiesis switch causes constitutive activation of MAPK in juvenile mice with Nf1 loss of heterozygosity (LOH); and (5) Nf1 LOH causes monocytosis in juvenile mice with Pten haploinsufficiency but does not cause lethality until adulthood. Our data suggest that 1 copy of Pten is sufficient to maintain an intact negative-feedback loop of the Akt pathway and HSC function in reconstitution, despite MAPK being constitutively activated in juvenile Pten+/DeltaNf1LOH mice. However, 2 copies of Pten are required to maintain the integrity of the MAPK pathway in juvenile mice with Nf1 haploinsufficiency. Our data indicate that previous investigations of Pten function in wild-type mice may not reflect the impact of Pten loss in mice with Nf1 mutations or other genetic defects. We provide a proof of concept that disassociated age-specific hematopoiesis contributes to leukemogenesis and pediatric demise.
View details for DOI 10.1182/bloodadvances.2020002326
View details for PubMedID 32777070
PTEN is indispensable for cells to respond to MAPK inhibitors in myeloid leukemia.
2018; 50: 72-79
Constitutively activated MAPK and AKT signaling pathways are often found in solid tumors and leukemias. PTEN is one of the tumor suppressors that are frequently found deficient in patients with late-stage cancers or leukemias. In this study we demonstrate that a MAPK inhibitor, PD98059, inhibits both AKT and ERK phosphorylation in a human myeloid leukemia cell line (TF-1), but not in PTEN-deficient leukemia cells (TF-1a). Ectopic expression of wild-type PTEN in myeloid leukemia cells restored cytokine responsiveness at physiological concentrations of GM-CSF (<0.02 ng/mL) and significantly improved cell sensitivity to MAPK inhibitor. We also found that Early Growth Response 1 (EGR1) was constitutively over-expressed in cytokine-independent TF-1a cells, and ectopic expression of PTEN down-regulated EGR1 expression and restored dynamics of EGR1 expression in response to GM-CSF stimulation. Data from primary bone marrow cells from mice with Pten deletion further supports that PTEN is indispensible for myeloid leukemia cells in response to MAPK inhibitors. Finally, We demonstrate that the absence of EGR1 expression dynamics in response to GM-CSF stimulation is one of the mechanisms underlying drug resistance to MAPK inhibitors in leukemia cells with PTEN deficiency. Our data suggest a novel mechanism of PTEN in regulating expression of EGR1 in hematopoietic cells in response to cytokine stimulation. In conclusion, this study demonstrates that PTEN is dispensable for myeloid leukemia cells in response to MAPK inhibitors, and PTEN regulates EGR1 expression and contributes to the cytokine sensitivity in leukemia cells.
View details for DOI 10.1016/j.cellsig.2018.06.004
View details for PubMedID 29964149
Timing of the loss of Pten protein determines disease severity in a mouse model of myeloid malignancy.
2016; 127 (15): 1912-22
Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric mixed myelodysplastic/myeloproliferative neoplasm (MDS/MPN). JMML leukemogenesis is linked to a hyperactivated RAS pathway, with driver mutations in the KRAS, NRAS, NF1, PTPN11, or CBL genes. Previous murine models demonstrated how those genes contributed to the selective hypersensitivity of JMML cells to granulocyte macrophage-colony-stimulating factor (GM-CSF), a unifying characteristic in the disease. However, it is unclear what causes the early death in children with JMML, because transformation to acute leukemia is rare. Here, we demonstrate that loss of Pten (phosphatase and tensin homolog) protein at postnatal day 8 in mice harboring Nf1 haploinsufficiency results in an aggressive MPN with death at a murine prepubertal age of 20 to 35 days (equivalent to an early juvenile age in JMML patients). The death in the mice was due to organ infiltration with monocytes/macrophages. There were elevated activities of protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) in cells at physiological concentrations of GM-CSF. These were more pronounced in mice with Nf1 haploinsufficiency than in littermates with wild-type Nf1,but this model is insufficient to cause cells to be GM-CSF hypersensitive. This new model represents a murine MPN model with features of a pediatric unclassifiable mixed MDS/MPN and mimics many clinical manifestations of JMML in terms of age of onset, aggressiveness, and organ infiltration with monocytes/macrophages. Our data suggest that the timing of the loss of PTEN protein plays a critical role in determining the disease severity in myeloid malignancies. This model may be useful for studying the pathogenesis of pediatric diseases with alterations in the Ras pathway.
View details for DOI 10.1182/blood-2015-05-646216
View details for PubMedID 26764354
View details for PubMedCentralID PMC4832508
The genomic landscape of juvenile myelomonocytic leukemia
2015; 47 (11): 1326-?
Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm (MPN) of childhood with a poor prognosis. Mutations in NF1, NRAS, KRAS, PTPN11 or CBL occur in 85% of patients, yet there are currently no risk stratification algorithms capable of predicting which patients will be refractory to conventional treatment and could therefore be candidates for experimental therapies. In addition, few molecular pathways aside from the RAS-MAPK pathway have been identified that could serve as the basis for such novel therapeutic strategies. We therefore sought to genomically characterize serial samples from patients at diagnosis through relapse and transformation to acute myeloid leukemia to expand knowledge of the mutational spectrum in JMML. We identified recurrent mutations in genes involved in signal transduction, splicing, Polycomb repressive complex 2 (PRC2) and transcription. Notably, the number of somatic alterations present at diagnosis appears to be the major determinant of outcome.
View details for DOI 10.1038/ng.3400
View details for PubMedID 26457647
Deficiency of CREB and over expression of miR-183 in juvenile myelomonocytic leukemia.
2013; 27 (7): 1585-8
View details for DOI 10.1038/leu.2013.49
View details for PubMedID 23417028
View details for PubMedCentralID PMC3715750
PTEN deficiency is a common defect in juvenile myelomonocytic leukemia.
2009; 33 (5): 671-7
The biological hallmark of juvenile myelomonocytic leukemia (JMML) is selective GM-CSF hypersensitivity. We hypothesized that PTEN protein deficiency might lead to insufficient negative growth signals to counter the hyperactive Ras signaling and therefore aid in the acceleration of the malignant transformation of JMML. In screening 34 JMML patients we found: (1) decreased PTEN protein in 67% of patients; (2) significantly lower PTEN mRNA levels in patients compared to controls (p<0.01); (3) a hypermethylated PTEN promoter in 77% of patients; and (4) constitutive-hyperactive Akt and MAPK in 55% and 73% of patients, respectively. These findings suggest that PTEN deficiency is very common in JMML and is in part due to hypermethylation of the PTEN gene promoter.
View details for DOI 10.1016/j.leukres.2008.09.036
View details for PubMedID 19010541
Rapid determination of clonality by detection of two closely-linked X chromosome exonic polymorphisms using allele-specific PCR.
The Journal of clinical investigation
1997; 99 (8): 1984-90
We reported two specific, reproducible, and quantitative clonality assays based on detection of exonic polymorphisms of the X chromosome genes p55 and G6PD using rtPCR-LDR. These assays are inconvenient for screening purposes. This study sought to develop a simple, reproducible assay, practical for screening genomic DNA samples for p55/G6PD genotypes, rapid clonality determination, and to determine the linkage relationship between these closely related loci. The salient feature of ASPCR is the performance of two PCR rounds. The first generates template; the second, using one aliquot of first-round products in two reaction tubes, each containing one allele-specific primer, detects each allele. ASPCR and rtPCR-LDR produced identical p55/G6PD results in 91 normal female genomic DNAs, and in 12 clonal hematopoietic disorder cDNAs, confirming assay validity. 209 female and 207 male genomic DNA samples were analyzed for p55/G6PD genotype by ASPCR; 60% of females were heterozygous at one or both loci. G6PD and p55 allelic frequencies were significantly different among African-American men and women, but were not significantly different among Caucasian men and women. These loci were in linkage equilibrium among African Americans, but not among Caucasians. ASPCR is a rapid, sensitive, and specific method for screening large numbers of genomic DNAs, and for rapid clonality determination.
View details for DOI 10.1172/JCI119366
View details for PubMedID 9109443
View details for PubMedCentralID PMC508023
Novel Humanized Loss-of-Function NF1 Mouse Model of Juvenile Myelomonocytic Leukemia
AMER SOC HEMATOLOGY. 2022: 9765-9766
View details for DOI 10.1182/blood-2022-167413
View details for Web of Science ID 000893230302333
Role of Cytokine Secretion Signatures of Donor-derived T Cells and Recipient Serum Cytokine Profiles as Predictive Biomarkers of Acute Graft-Versus-Host Disease in alpha beta T-cell/CD19 B-cell Depleted Hematopoietic Stem Cell Transplant Pediatric Recipients
View details for Web of Science ID 000788322300433
- POTENTIAL BIOMARKERS OF ACUTE GVHD IN ALPHA/BETA T-CELL/B-CELL DEPLETED HSCT PEDIATRIC RECIPIENTS WILEY. 2022
Aberrant Histone Landscape in Juvenile Myelomonocytic Leukemia
AMER SOC HEMATOLOGY. 2021
View details for DOI 10.1182/blood-2021-152074
View details for Web of Science ID 000835740101069
Combinatorial Cytokine Secretion Signature of Donor-Derived T Cells Infused with the Graft: A New Potential Biomarker of Acute Graft-Versus-Host Disease in.ss t-Cell/CD19 B-Cell Depleted Hematopoietic Stem Cell Transplant Recipients
AMER SOC HEMATOLOGY. 2021
View details for DOI 10.1182/blood-2021-149948
View details for Web of Science ID 000736398806151
M1 and M2 macrophages differentially regulate hematopoietic stem cell self-renewal and ex vivo expansion.
2018; 2 (8): 859-870
Uncovering the cellular and molecular mechanisms by which hematopoietic stem cell (HSC) self-renewal is regulated can lead to the development of new strategies for promoting ex vivo HSC expansion. Here, we report the discovery that alternative (M2)-polarized macrophages (M2-MΦs) promote, but classical (M1)-polarized macrophages (M1-MΦs) inhibit, the self-renewal and expansion of HSCs from mouse bone marrow (BM) in vitro. The opposite effects of M1-MΦs and M2-MΦs on mouse BM HSCs were attributed to their differential expression of nitric oxide synthase 2 (NOS2) and arginase 1 (Arg1), because genetic knockout of Nos2 and Arg1 or inhibition of these enzymes with a specific inhibitor abrogated the differential effects of M1-MΦs and M2-MΦs. The opposite effects of M1-MΦs and M2-MΦs on HSCs from human umbilical cord blood (hUCB) were also observed when hUCB CD34+ cells were cocultured with M1-MΦs and M2-MΦs generated from hUCB CD34- cells. Importantly, coculture of hUCB CD34+ cells with human M2-MΦs for 8 days resulted in 28.7- and 6.6-fold increases in the number of CD34+ cells and long-term SCID mice-repopulating cells, respectively, compared with uncultured hUCB CD34+ cells. Our findings could lead to the development of new strategies to promote ex vivo hUCB HSC expansion to improve the clinical utility and outcome of hUCB HSC transplantation and may provide new insights into the pathogenesis of hematological dysfunctions associated with infection and inflammation that can lead to differential macrophage polarization.
View details for DOI 10.1182/bloodadvances.2018015685
View details for PubMedID 29666049
View details for PubMedCentralID PMC5916005
Metabolic history impacts mammary tumor epithelial hierarchy and early drug response in mice.
2016; 23 (9): 677-90
The emerging links between breast cancer and metabolic dysfunctions brought forth by the obesity pandemic predict a disproportionate early disease onset in successive generations. Moreover, sensitivity to chemotherapeutic agents may be influenced by the patient's metabolic status that affects the disease outcome. Maternal metabolic stress as a determinant of drug response in progeny is not well defined. Here, we evaluated mammary tumor response to doxorubicin in female mouse mammary tumor virus-Wnt1 transgenic offspring exposed to a metabolically compromised environment imposed by maternal high-fat diet. Control progeny were from dams consuming diets with regular fat content. Maternal high-fat diet exposure increased tumor incidence and reduced tumor latency but did not affect tumor volume response to doxorubicin, compared with control diet exposure. However, doxorubicin-treated tumors from high-fat-diet-exposed offspring demonstrated higher proliferation status (Ki-67), mammary stem cell-associated gene expression (Notch1, Aldh1) and basal stem cell-like (CD29(hi)CD24(+)) epithelial subpopulation frequencies, than tumors from control diet progeny. Notably, all epithelial subpopulations (CD29(hi)CD24(+), CD29(lo)CD24(+), CD29(hi)CD24(+)Thy1(+)) in tumors from high-fat-diet-exposed offspring were refractory to doxorubicin. Further, sera from high-fat-diet-exposed offspring promoted sphere formation of mouse mammary tumor epithelial cells and of human MCF7 cells. Untargeted metabolomics analyses identified higher levels of kynurenine and 2-hydroxyglutarate in plasma of high-fat diet than control diet offspring. Kynurenine/doxorubicin co-treatment of MCF7 cells enhanced the ability to form mammosphere and decreased apoptosis, relative to doxorubicin-only-treated cells. Maternal metabolic dysfunctions during pregnancy and lactation may be targeted to reduce breast cancer risk and improve early drug response in progeny, and may inform clinical management of disease.
View details for DOI 10.1530/ERC-16-0136
View details for PubMedID 27402613
View details for PubMedCentralID PMC4997088
Phase II/III trial of a pre-transplant farnesyl transferase inhibitor in juvenile myelomonocytic leukemia: a report from the Children's Oncology Group.
Pediatric blood & cancer
2015; 62 (4): 629-36
Juvenile myelomonocytic leukemia (JMML) is not durably responsive to chemotherapy, and approximately 50% of patients relapse after hematopoietic stem cell transplant (HSCT). Here we report the activity and acute toxicity of the farnesyl transferase inhibitor tipifarnib, the response rate to 13-cis retinoic acid (CRA) in combination with cytoreductive chemotherapy, and survival following HSCT in children with JMML.Eighty-five patients with newly diagnosed JMML were enrolled on AAML0122 between 2001 and 2006. Forty-seven consented to receive tipifarnib in a phase II window before proceeding to a phase III trial of CRA in combination with fludarabine and cytarabine followed by HSCT and maintenance CRA. Thirty-eight patients enrolled only in the phase III trial.Overall response rate was 51% after tipifarnib and 68% after fludarabine/cytarabine/CRA. Tipifarnib did not increase pre-transplant toxicities. Forty-six percent of the 44 patients who received protocol compliant HSCT relapsed. Five-year overall survival was 55 ± 11% and event-free survival was 41 ± 11%, with no significant difference between patients who did or did not receive tipifarnib.Administration of tipifarnib in the window setting followed by HSCT in patients with newly diagnosed JMML was safe and yielded a 51% initial response rate as a single agent, but failed to reduce relapse rates or improve long-term overall survival.
View details for DOI 10.1002/pbc.25342
View details for PubMedID 25704135
View details for PubMedCentralID PMC4339233
Subclonal mutations in SETBP1 confer a poor prognosis in juvenile myelomonocytic leukemia.
2015; 125 (3): 516-24
Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of childhood associated with a poor prognosis. Recently, massively parallel sequencing has identified recurrent mutations in the SKI domain of SETBP1 in a variety of myeloid disorders. These lesions were detected in nearly 10% of patients with JMML and have been characterized as secondary events. We hypothesized that rare subclones with SETBP1 mutations are present at diagnosis in a large portion of patients who relapse, but are below the limits of detection for conventional deep sequencing platforms. Using droplet digital polymerase chain reaction, we identified SETBP1 mutations in 17/56 (30%) of patients who were treated in the Children's Oncology Group sponsored clinical trial, AAML0122. Five-year event-free survival in patients with SETBP1 mutations was 18% ± 9% compared with 51% ± 8% for those without mutations (P = .006).
View details for DOI 10.1182/blood-2014-09-601690
View details for PubMedID 25395418
View details for PubMedCentralID PMC4296011
Mutations in GATA2 are rare in juvenile myelomonocytic leukemia.
2014; 123 (9): 1426-7
View details for DOI 10.1182/blood-2013-11-531079
View details for PubMedID 24578498
View details for PubMedCentralID PMC3938154
PTEN transcript variants caused by illegitimate splicing in "aged" blood samples and EBV-transformed cell lines.
2010; 128 (6): 609-14
PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. Mutations occur in either heritable or sporadic fashion. Sequencing of cDNA from patients and normal individuals often reveals splicing variants (SVs) of PTEN, some of which are non-mutation related. To investigate whether these SVs were the result of illegitimate splicing (a general decrease of fidelity in splicing site selection in "aged" samples), we tested "aged" blood from individuals who had normal PTEN transcripts in their "fresh" mononuclear cells. Blood from 20 normal individuals was collected and split into two aliquots. Total RNA and DNA were extracted immediately ("fresh") and 48 h later ("aged"), respectively. Using RT-PCR, subcloning and sequencing, we found seven types of SVs. No mutation was detected in the related intron-exon flanking region in genomic DNA in either "fresh" or "aged" samples. Some of the SVs were also consistently present in both the "fresh" and "aged" EBV-transformed lymphoblastoid cells from six normal individuals. Western blot data indicated that the PTEN protein level (in full length) was not altered in the "fresh" EBV-transformed lymphoblastoid cells with SVs. In conclusion, our data demonstrate that PTEN illegitimate splicing often occurs in "aged" blood and EBV-transformed lymphoblastoid cells. Therefore, it is critical to note the time point of RNA extraction when investigating for PTEN aberrant transcripts. We hope that our data will increase awareness about the sample status, because gene expression data may be potentially flawed from "aged" samples, particularly when dealing with clinical samples.
View details for DOI 10.1007/s00439-010-0886-4
View details for PubMedID 20839010
View details for PubMedCentralID PMC2978886
mrtl-A translation/localization regulatory protein encoded within the human c-myc locus and distributed throughout the endoplasmic and nucleoplasmic reticular network.
Journal of cellular biochemistry
2008; 105 (4): 1092-108
mrtl (myc-related translation/localization regulatory factor) is a previously uncharacterized protein synthesized from the first open reading frame contained within the human c-myc P0 transcript, approximately 800 nucleotides upstream of the Myc coding sequence. The mrtl protein, 114 amino acids in length, is projected to contain an N-terminal transmembrane domain and a highly charged C-terminal interaction domain with homology to numerous RNA-binding proteins. Using monoclonal antibodies raised against the hydrophilic C-terminal domain, endogenous mrtl was visualized in human breast tumor cell lines and primary mammary epithelial cells at the nuclear envelope and contiguous endoplasmic/nucleoplasmic reticulum. mrtl colocalizes and coimmunoprecipitates with translation initiation factor eIF2alpha and the 40S ribosomal protein RACK1, and appears capable of binding specifically to the c-myc RNA. Inducible ectopic overexpression of wild-type mrtl interferes with the function of endogenous mrtl, which results in loss of Myc from the nucleus. Furthermore, treatment of cells with a peptide derived from the C-terminal domain displaces endogenous mrtl and causes a dramatic reduction in total cellular Myc protein levels. Together with our previous work demonstrating complete loss of tumorigenicity in association with ectopic expression of the c-myc P0 5'-UTR (containing the mrtl coding sequence), these results suggest that mrtl may serve an important function in regulating Myc translation and localization to the nucleus, perhaps ultimately contributing to the role of the c-myc locus in oncogenesis.
View details for DOI 10.1002/jcb.21909
View details for PubMedID 18816594
View details for PubMedCentralID PMC2587529
Activating FLT3 mutations are rare in children with juvenile myelomonocytic leukemia.
Pediatric blood & cancer
2005; 44 (2): 142-6
Activating mutations of FLT3 have been identified in multiple myeloid malignancies. Two types of activating mutations have been described: (1) the internal tandem duplication (FLT3-ITD) and (2) point mutations within the activating loop (FLT3-ALM). Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative disorder of early childhood. Mutations and other genetic abnormalities of RAS, NF1, and PTPN11 have been implicated as causative events in JMML, but approximately 25% of JMML patients harbor none of these abnormalities. We investigated whether FLT3 mutations might also contribute to JMML pathogenesis, and if present, whether FLT3 status would correlate with disease natural history and prognosis.Genomic DNA was isolated from peripheral blood and bone marrow samples of 60 patients meeting international JMML diagnostic criteria. Samples were analyzed for FLT3-ITD and FLT3-ALM using polymerase chain reaction and restriction endonuclease digestion.FLT3-ALM was found in 1/60 (1.7%) patients analyzed. Direct sequencing confirmed a C836G mutation. Clinical and laboratory characteristics of the JMML patient with the FLT3-ALM did not differ from the remainder of the cohort. No FLT3-ITD mutations were detected.This first reported mutational analysis for both FLT3-ITD and FLT3-ALM performed in JMML documents the presence of FLT3 mutations within JMML, but at a sufficiently low prevalence as to be clinically insignificant for most patients. Despite the poor prognosis and limited therapeutic options for JMML patients with refractory disease, compassionate therapy with targeted FLT3 inhibitors should not be considered in this patient population until adequate safety and efficacy data become available.
View details for DOI 10.1002/pbc.20176
View details for PubMedID 15390271
Regulation of ferrochelatase gene expression by hypoxia.
2004; 75 (17): 2035-43
Ferrochelatase (FECH), the last enzyme of the heme biosynthetic pathway, catalyzes the insertion of iron into protoporphyrin to form heme. This pathway provides heme for hemoglobin and other essential hemoproteins. The regulatory role of oxygen in the pathway has not been clearly established. In this study, we examined whether FECH gene expression is upregulated during hypoxia by a mechanism which involves the hypoxia-inducible factor 1 (HIF-1). Two HIF-1 binding motifs were identified within the -150 bp FECH minimal promoter sequence. Exposure of HEL, K562, and Hep-G2 cells to hypoxia for 18 hours resulted in a significant increase in FECH mRNA expression (p < 0.05). Hypoxia also transactivated the minimal promoter for the FECH gene in the cells. Transient co-expression of wild-type HIF-1alpha or a dominant negative HIF-1alpha with the FECH minimal promoter luciferase construct stimulated or blocked FECH promoter activity, respectively. Expression of the von Hippel-Lindau (VHL) tumor suppressor factor blocked the expression of both FECH mRNA and HIF-1alpha protein during normoxic culture of renal carcinoma cell line (RCC4). The results suggest that the FECH gene is a target for HIF-1 during hypoxia.
View details for DOI 10.1016/j.lfs.2004.03.027
View details for PubMedID 15312748
Ferrochelatase gene mutations in erythropoietic protoporphyria: focus on liver disease.
Cellular and molecular biology (Noisy-le-Grand, France)
2002; 48 (1): 83-9
A deficiency of ferrochelatase (FECH) activity underlies the excess accumulation of protoporphyrin that occurs in erythropoietic protoporphyria (EPP). In some patients, protoporphyrin accumulation causes liver damage that necessitates liver transplantation. The purpose of this study was to determine if specific mutations in the FECH gene are present in patients who develop liver disease. FECH cDNA and all 11 exons and their flanking intron regions in the FECH gene were amplified and sequenced by specific polymerase chain reactions. Gene mutations were determined in 34 individuals from 24 families: 14 had liver disease, 10 necessitating liver transplantation. All individuals were heterozygous for mutations that altered the coding region of FECH mRNA. The mutations in patients with liver disease were heterogenous, but usually caused a major structural alterations in the FECH protein, most commonly as a result of exon skipping in FECH mRNA. However, the mutations could not account for the severe phenotype by themselves, since the same mutations were found in asymptomatic family members of patients with liver disease and in patients from families in which liver disease was not present. Other genetic factors, and possibly acquired factors, also must be critical to the development of this severe phenotype in EPP.
View details for PubMedID 11929052
Unusual patterns of exon skipping in Bruton tyrosine kinase are associated with mutations involving the intron 17 3' splice site.
American journal of human genetics
1997; 60 (4): 798-807
Seven individuals with the diagnosis of X-linked agammaglobulinemia were analyzed for mutations in Bruton tyrosine kinase (Btk) gene at both the cDNA transcript and genomic DNA levels. In addition, maternal carrier status was determined in six of the seven families by examining X chromosome-inactivation patterns for B cells in comparison with other types of blood cells. Three categories of mutations were identified: (1) three patients have missense mutations in either the pleckstrin or SH2 domains of Btk; (2) three patients exhibit mutations at or near intron/exon splice sites, two of which represent inherited mutations within the kinase domain; and (3) one patient has inherited a 2.5-kb deletion with the loss of a DNA segment encoding three exons of the kinase domain. Variation in the lengths of Btk transcripts was evident in two patients with splice-site mutations and in the patient with the DNA deletion. Sequences of the different cDNA transcripts from the patients with 3' splice-site mutations reveal complex patterns of exon skipping involving from one to four exons of the kinase domain. These findings implicate 3' splice sites of the penultimate exon in the recognition or processing of upstream exons.
View details for PubMedID 9106525
View details for PubMedCentralID PMC1712487
Maternal infusion of antioxidants (Trolox and ascorbic acid) protects the fetal heart in rabbit fetal hypoxia.
1996; 39 (3): 499-503
The antioxidants, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, a water soluble analog of vitamin E) and ascorbic acid (AA), protect the heart from ischemia-reperfusion injury. We hypothesized that maternal infusion of Trolox and AA, would reduce the fetal bradycardia and myocardial damage observed in fetal hypoxia and increase the total antioxidant activity in fetal plasma. Either i.v. saline (control group) or Trolox + AA (drug group) was randomly administered to 29-d-old pregnant rabbits. Fetal hypoxia was induced by uterine ischemia. Fetal heart rate, plasma CK-MB activity, and plasma total radical antioxidant potential (TRAP) were measured in different sets of animals. Fetal heart rate in the drug group was higher than in the control group for the first 35 min (p < 0.05 at every 5-min interval). Fetal bradycardia (<60 beats/min) occurred after 39 min (median) in the drug group, and 29 min in the control group (p < 0.05). After 50 min of hypoxia, plasma CK-MB was lower in the drug group, 1204 +/- 132 U/L (mean +/- SEM), than in the control group, 2633 +/- 233 U/L (p < 0.05). TRAP was higher in the drug group, 3.01 +/- 0.15 mM (Trolox equivalent concentration), than in the control group, 1.48 +/- 0.27 mM (p < 0.05). Higher TRAP levels (> or = 2.0 mM) were associated with lower CK-MB levels (<2500 U/L) (p < 0.05). Administration of Trolox and AA to the mother has a beneficial effect on fetal myocardial damage after fetal hypoxia, and a small beneficial effect on fetal bradycardia during hypoxia. The beneficial effect may be due to the augmentation of fetal plasma antioxidants from maternal antioxidant pretreatment.
View details for DOI 10.1203/00006450-199603000-00019
View details for PubMedID 8929872
A novel clonality assay based on transcriptional polymorphism of X chromosome gene p55.
Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation
1995; 1 (2): 81-7
The clonal nature of cell populations in malignant and myeloproliferative disorders can be determined in female subjects by random-inactivation assays of the X chromosome. Assays utilizing either expression of the G6PD isozymes or DNA-methylation differences between the active and inactive X chromosomes have significant short-comings. We developed a test based on nucleotide #1311 exonic polymorphism of G6PD that allows detection of clonality by determining the transcriptional polymorphism of the active X chromosome using a reverse transcription-polymerase chain reaction-ligase detection reaction (rtPCR-LDR). Since only 18% of females in the United States are informative (heterozygous) for this chromosome with this assay, we searched for other exonic X chromosome polymorphisms. Concentrating on a discrepancy (G or T at cDNA #358) of published sequences of ubiquitously expressed gene, palmitoylated membrane protein for p55, we confirmed this conservative polymorphism at the cDNA level. To detect the genotype of this polymorphism, we established the intron/exon boundary of the first 5' exons and determined the whole sequence of the second intron. We found that the polymorphic site is at the third exon, nine nucleotides downstream from the 294-bp second intron. This close proximity to the intron necessitated the development of a separate PCR-LDR reaction with oligonucleotides for cDNA and genomic DNA. Furthermore, we determined that the p55 gene is subject to X chromosome inactivation. Based on these observations, we developed a novel p55 clonality assay that is reproducible, quantitative, and very sensitive. Screening of 37 randomly selected healthy females of Caucasian, African-American, and Asian origin revealed that 38% of females are informative when this assay is used. We demonstrate a multiple crossover between the G6PD and the p55 polymorphisms (separated by approximately 200 kb), suggesting that these two polymorphisms are in linkage disequilibrium; thus, approximately 50% of female subjects are informative for clonality studies using the two assays.
View details for PubMedID 9118296