A Patient with Kabuki Syndrome Mutation Presenting with Very Severe Aplastic Anemia.

Kabuki syndrome (KS) is a rare congenital disorder commonly complicated by humoral immunodeficiency. Patients with KS present with mutation in the histone-lysine N-methyltransferase 2D (KMT2D) gene. Although various KMT2D mutations are often identified in lymphoma and leukemia, those encountered in aplastic anemia (AA) are limited. Herein, we present the case of a 45-year-old Japanese man who developed severe pancytopenia and hypogammaglobulinemia. He did not present with any evident malformations, intellectual disability, or detectable levels of autoantibodies. However, B-cell development was impaired. Therefore, a diagnosis of very severe AA due to a hypoplastic marrow, which did not respond to granulocyte colony-stimulating factor, was made. The patient received umbilical cord blood transplantation but died from a Pseudomonas infection before neutrophil engraftment. Trio whole-exome sequencing revealed a novel missense heterozygous mutation c.15959G >A (p.R5320H) in exon 50 of the KMT2D gene. Moreover, Sanger sequencing of peripheral blood and bone marrow mononuclear cells and a skin biopsy specimen obtained from this patient identified this heterozygous mutation, suggesting that de novo mutation associated with KS occurred in the early embryonic development. Our case showed a novel association between KS mutation and adult-onset AA.

T-cell expression of Bruton's tyrosine kinase promotes autoreactive T-cell activation and exacerbates aplastic anemia.

The role of Bruton's tyrosine kinase (BTK) in BCR signaling is well defined, and BTK is involved in B-cell development, differentiation, and malignancies. However, the expression of Btk in T cells and its role in T-cell function remain largely unknown. Here, we unexpectedly found high expression and activation of BTK in T cells. Deficiencies in BTK resulted in the impaired activation and proliferation of autoreactive T cells and ameliorated bone marrow failure (BMF) in aplastic anemia. Mechanistically, BTK is activated after TCR engagement and then phosphorylates PLCγ1, thus promoting T-cell activation. Treatment with acalabrutinib, a selective BTK inhibitor, decreased T-cell proliferation and ameliorated BMF in mice with aplastic anemia. Our results demonstrate an unexpected role of BTK in optimal T-cell activation and in the pathogenesis of autoimmune aplastic anemia, providing insights into the molecular regulation of T-cell activation and the pathogenesis of T-cell-mediated autoimmune disease.

PKM2 Is Required to Activate Myeloid Dendritic Cells from Patients with Severe Aplastic Anemia.

Severe aplastic anemia (SAA) is an autoimmune disease in which bone marrow failure is mediated by activated myeloid dendritic cells (mDCs) and T lymphocytes. Recent research has identified a strong immunomodulatory effect of pyruvate kinase M2 (PKM2) on dendritic cells in immune-mediated diseases. In this study, we aimed to explore the role of PKM2 in the activation of mDCs in SAA. We observed conspicuously higher levels of PKM2 in mDCs from SAA patients compared to normal controls at both the gene and protein levels. Concurrently, we unexpectedly discovered that after the mDC-specific downregulation of PKM2, mDCs from patients with SAA exhibited weakened phagocytic activity and significantly decreased and shortened dendrites relative to their counterparts from normal controls. The expression levels of the costimulatory molecules CD86 and CD80 were also reduced on mDCs. Our results also suggested that PKM2 knockdown in mDCs reduced the abilities of these cells to promote the activation of CD8+ T cells (CTLs), leading to the decreased secretion of cytotoxic factors by the latter cell type. These findings demonstrate that mDC activation requires an elevated intrinsic PKM2 level and that PKM2 improves the immune status of patients with SAA by enhancing the functions of mDCs and, consequently, CTLs.

Aldehyde dehydrogenase 2 in aplastic anemia, Fanconi anemia and hematopoietic stem cells.

Maintenance of the hematopoietic stem cell (HSC) compartment depends on the ability to metabolize exogenously and endogenously generated toxins, and to repair cellular damage caused by such toxins. Reactive aldehydes have been demonstrated to cause specific genotoxic injury, namely DNA interstrand cross-links. Aldehyde dehydrogenase 2 (ALDH2) is a member of a 19 isoenzyme ALDH family with different substrate specificities, subcellular localization, and patterns of expression. ALDH2 is localized in mitochondria and is essential for the metabolism of acetaldehyde, thereby placing it directly downstream of ethanol metabolism. Deficiency in ALDH2 expression and function are caused by a single nucleotide substitution and resulting amino acid change, called ALDH2*2. This genetic polymorphism affects 35-45% of East Asians (about ~560 million people), and causes the well-known Asian flushing syndrome, which results in disulfiram-like reactions after ethanol consumption. Recently, the ALDH2*2 genotype has been found to be associated with marrow failure, with both an increased risk of sporadic aplastic anemia and more rapid progression of Fanconi anemia. This review discusses the unexpected interrelationship between aldehydes, ALDH2 and hematopoietic stem cell biology, and in particular its relationship to Fanconi anemia.

Deregulation of vital mitotic kinase-phosphatase signaling in hematopoietic stem/progenitor compartment leads to cellular catastrophe in experimental aplastic anemia.

Aplastic anemia, the paradigm of bone marrow failure, is characterized by pancytopenic peripheral blood and hypoplastic bone marrow. Among various etiologies, inappropriate use of DNA alkylating drugs like cyclophosphamide and busulfan often causes the manifestation of the dreadful disease. Cell cycle impairment in marrow hematopoietic stem/progenitor compartment together with cellular apoptosis has been recognized as culpable factors behind aplastic pathophysiologies. However, the intricate molecular mechanisms remain unrevealed till date. In the present study, we have dealt with the mechanistic intervention of the disease by peripheral blood hemogram, bone marrow histopathology, cytopathology, hematopoietic kinetic study, scanning electron microscopy, DNA damage assessment and flowcytometric analysis of cellular proliferation and apoptosis in hematopoietic stem/progenitor cell (HSPC) rich marrow compartment using busulfan and cyclophosphamidemediated mouse model. To unveil the molecular mechanisms behind aplastic pathophysiology, we further investigated the role of some crucial mitotic and apoptotic regulators like Protein kinase-B (PKB), Gsk-3ß, Cyclin-D1, PP2A, Cdc25c, Plk-1, Aurora kinase-A, Chk-1 regarding the hematopoietic catastrophe. Our observations revealed that the alteration of PKB-GSK-3ß axis, Plk-1, and Aurora kinase-A expressions in HSPC compartment due to DNA damage response was associated with the proliferative impairment and apoptosis during aplastic anemia. The study established the correlation between the accumulation of DNA damage and alteration of the mentioned molecules in aplastic HSPCs that lead to the hematopoietic catastrophe. We anticipate that our findings will be beneficial for developing better therapeutic strategies for the dreadful disease concerned.

Association of GSTM1 and GSTT1 deletion polymorphisms with Pakistani aplastic anemia patients and controls and meta-analysis.

Interaction of environmental and genetic elements plays a vital role in the pathogenesis of aplastic anemia (AA). Glutathione S-transferase (GST) is a key detoxifying enzyme. Absence or low levels of this enzyme may genetically predispose individuals to AA. GST genes GSTM1 and GSTT1 are polymorphic. The aim of this study was to screen Pakistani AA patients and controls for GSTM1 deletion GSTM0 and GSTT1 deletion GSTT0 and perform meta-analysis using our data and other published data regarding these polymorphisms. DNA samples from 137 patients and 220 controls were screened using multiplex polymerase chain reaction. GSTM0 emerged as susceptible genotype for AA in Pakistan with a percentage frequency of 49.6 % as compared to 30 % in controls with odds ratio (OR) of 2.25, 95 % confidence interval (CI) of 1.4-3.5 and corrected p = 0.006. The meta-analysis showed a significant association between the null genotype GSTT0 and AA in overall analysis with OR of 1.47, 95 % CI of 1.01-2.13 and p value of 0.04 in random effects model. Studies like these could play a role in understanding the underlying path in AA pathogenesis and therefore can help in designing means for prevention, diagnose and treatment.

Busuishengxue ranules mediate their effects upon non-severe aplastic anemia via mitogen-activated protein kinase/extracellular signal-regulated kinase pathway.

OBJECTIVE: To observe the clinical efficacy of Busuishengxue granules on non-severe aplastic anemia (NSAA) and investigate its effect on the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. METHODS: Sixty NSAA patients were divided equally into two groups. Subjects in the experimental group were treated with Busuishengxue granules, and the control group with Zaizaoshengxue tablets. The treatment course was 6 months and curative efficacy was compared between the two groups as well as with 10 healthy individuals. Flow cytometry (FCM) was used to detect the intracellular concentration of Ca2+ ([Ca2+]i). Western blotting was employed to detect the expression of enzymes in the MAPK/ERK pathway. RESULTS: The efficacy of Busuishengxue granules was significantly better than that of Zaizaoshengxue tablets (P < 0.05). Before treatment, expression of JNK, phospho-ERK 1/2 and p-JNK was higher, and [Ca2+]i higher, than that of the control group (P < 0.05). After treatment with Busuishengxue granules, expression of all enzymes related to signal transduction pathways in the blood cells of NSSA patients were altered to different degrees. CONCLUSION: Busuishengxue granules had a better effect with regard to improving symptom scores, increasing the number of blood leukocytes, and increasing hemoglobin levels than Zaizaosh-engxue tablets, and they differed slightly in terms of increasing the number of platelets.

[The relationship of telomere and telomerase activity with outcome of aplastic anemia after immunosuppressive therapy].

OBJECTIVE: To observe the changes of telomere length and telomerase activity in patients with aplastic anemia (AA), and relationship with immunosuppressive therapy (IST) efficacy, to explore the pathogenesis of AA and the role of telomere length in evaluating immunosuppressive therapy efficacy. METHODS: 71 cases of AA patients between September 2010 and March 2013 were enrolled into this study. 3 ml peripheral blood specimens from this cohort of patients were collected to test the telomere length in peripheral blood mononuclear cell (PBMNC) with flow-FISH and detect telomerase activity with TRAP-PCR-ELISA method. RESULTS: Telomere length and age showed negative correlation (b=-0.387, P=0.001) in normal control, NSAA and SAA + VSAA groups, telomere length became shorter with the growth of age, and normal control group telomere length decreased along with the age growth slightly greater than the other two groups (NSAA, SAA+VSAA). Besides the effect of age on telomere length, no significant difference was observed between NSAA and SAA+VSAA groups (P=0.573), and NSAA, SAA+VSAA (30.957 ± 4.502,29.510 ± 5.911)groups were significantly shorter than normal control group (51.086±10.844) (P<0.01). Telomere length in NR group (25.357±4.848)was significantly lower than normal control group (51.086 ± 10.844) (P=0.005), telomere length in CR(32.808 ± 4.685)/PR groups (30.334±4.464) compared with normal control group had no significant difference (P=0.517, P=0.254). Telomere length below 29.21% obviously decreased outcomes of IST. Telomerase activity had significant difference (χ²=20.385, P<0.01). The telomerase activity had no significant difference in terms of age and gender in three groups, multiple comparison found that telomerase activities in SAA + VSAA (0.324±0.178) (P<0.01), and NSAA (0.234±0.175) groups (P=0.002) were significantly higher than normal control group (0.107±0.083). CONCLUSION: Telomere length of PBMNC in AA patients was significantly shortened than normal control group with telomerase activity increased, and telomere shorted more apparently in NR group, these patients should adjust the treatment as early as possible. Telomeres could predict the curative effect of IST.

Common polymorphic deletion of glutathione S-transferase theta predisposes to acquired aplastic anemia: Independent cohort and meta-analysis of 609 patients.

Acquired aplastic anemia (AA) is a rare life-threatening bone marrow failure syndrome, caused by autoimmune destruction of hematopoietic stem and progenitor cells. Epidemiologic studies suggest that environmental exposures and metabolic gene polymorphisms contribute to disease pathogenesis. Several case-control studies linked homozygous deletion of the glutathione S-transferase theta (GSTT1) gene to AA; however, the role of GSTT1 deletion remains controversial as other studies failed to confirm the association. We asked whether a more precise relationship between the GSTT1 null polymorphism and aplastic anemia could be defined using a meta-analysis of 609 aplastic anemia patients, including an independent cohort of 67 patients from our institution. We searched PubMed, Embase, and the Cochrane Database for studies evaluating the association between GSTT1 null genotype and development of AA. Seven studies, involving a total of 609 patients and 3,914 controls, fulfilled the eligibility criteria. Meta-analysis revealed a significant association of GSTT1 null genotype and AA, with an OR = 1.74 (95% CI 1.31-2.31, P < 0.0001). The effect was not driven by any one individual result, nor was there evidence of significant publication bias. The association between AA and GSTT1 deletion suggests a role of glutathione-conjugation in AA, possibly through protecting the hematopoietic compartment from endogenous metabolites or environmental exposures. We propose a model whereby protein adducts generated by reactive metabolites serve as neo-epitopes to trigger autoimmunity in aplastic anemia.

[Expression of telomerase activity and its related genes in the marrow hemopoietic stem cells of children with aplastic anemia].

OBJECTIVE: To explore the relationship of telomerase RNA component (hTERC) and the telomerase reverse transcriptase (hTERT) with telomerase activity in the marrow hemopoietic stem cells of children with aplastic anemia (AA). METHODS: Fifty-two children with chronic AA, 13 children with acute AA and 21 normal controls were enrolled in the study. Telomerase activity and the expression of mRNA of hTERT and hTERC were detected by Telomeric Repeat Amplification Protocol (TRAP) with silver staining and real-time Q-PCR respectively. RESULTS: Levels of telomerase activity in both the chronic and acute AA groups were higher than in the control group (P<0.01). The AA groups had significantly higher expression of hTERT mRNA than the control group (P<0.01). The chronic AA group had higher expression of hTERT mRNA and telomerase activity than the acute AA group (P<0.05). There was no significant difference in the expression of hTERC mRNA among the three groups (P=0.812). There was a significant correlation between the expression of hTERT mRNA and telomerase activity (r=0.660, P<0.01). CONCLUSIONS: Expression of telomerase activity may be involved in the pathophysiology and development of AA, and hTERT plays a crucial role in expression of telomerase activity.

Genetic polymorphisms of glutathione-S-transferase and microsomal epoxide hydrolase in egyptian acquired aplastic anemia patients.

Exposure to various environmental toxins with a reduced ability to metabolize them may lead to acquired aplastic anemia (AA). Genetic polymorphism of the detoxifying enzymes, the glutathione-S-transferase (GST) and microsomal epoxide hydrolase (mEh), with alteration in their activities could explain the genetic interindividual risks for AA. We aimed to characterize the genetic polymorphisms of the GST and mEh and to test their impact on the susceptibility, disease severity, and prognosis in Egyptian patients with AA. The GST and mEh genotypes were determined by multiplex-polymerase chain reaction and polymerase chain reaction-restriction fragment length polymorphism analysis, respectively, in 21 patients with AA and 20 healthy control subjects. The mEh functional phenotypes were assessed. The frequency of GST θ1-null genotype was found significantly higher in AA patients compared with the controls (odds ratio=2.8, 95% confidence interval = 1.1-7.8; P = 0.001). The frequency of heterozygous 139A--G of the mEh gene was significantly higher in AA patients compared with the controls (odds ratio=3.07, 95% confidence interval = 1.23-7.7; P = 0.018). Moreover, the patients with normal functional phenotype of the mEh had significantly favorable prognosis than those with abnormal enzyme activity (P = 0.027). Thus, the GST θ1-null genotype and the 139A--G mEh gene polymorphism may enhance the susceptibility to AA and provide an evidence of gene-environmental interaction.

Telomerase gene mutation screening in Chinese patients with aplastic anemia.

To study the incidence of telomerase gene mutations in Chinese patients with acquired bone marrow failure (BMF) and explore its relationship with telomere shortening. Blood samples from 66 patients with aplastic anemia (AA) in northern China were collected and TERC mutation analysis was performed. Two TERC mutations were identified. The incidence of telomerase gene mutations in Chinese people with acquired AA is similar to that of the western people.

[Lipid peroxidation and activity of antioxidative protection enzymes in patients with aplastic anemia].

Aplastic anaemia (AA) in connection with liver damage is characterized by severe course of the disease, significant shift of the myelogram, considerable changes of blood picture, high lethality, taking in this pathological process various organs and digestion systems. Results of ultrasonic and biochemical researches show presence of deep changes in the liver, gall bladder and pancreas. The disbalance of lipid peroxidation and antioxidative protection system have been observed in these patients. It is revealed by decrease in the activity of enzymes of superoxide dismutase, glutathione peroxidase and catalase, in increase in capacity of intermediate and end products of POL. It is especially expressed in combination of AA with liver damage. That's why it is necessary to provide a constant control of these organs.

Sex hormones, acting on the TERT gene, increase telomerase activity in human primary hematopoietic cells.

Androgens have been used in the treatment of bone marrow failure syndromes without a clear understanding of their mechanism of action. Blood counts of patients with dyskeratosis congenita or aplastic anemia with mutations in telomerase genes can improve with androgen therapy. Here we observed that exposure in vitro of normal peripheral blood lymphocytes and human bone marrow-derived CD34(+) cells to androgens increased telomerase activity, coincident with higher TERT mRNA levels. Cells from patients who were heterozygous for telomerase mutations had low baseline telomerase activity, which was restored to normal levels by exposure to androgens. Estradiol had an effect similar to androgens on TERT gene expression and telomerase enzymatic activity. Tamoxifen abolished the effects of both estradiol and androgens on telomerase function, and letrozole, an aromatase inhibitor, blocked androgen effects on telomerase activity. Conversely, flutamide, an androgen receptor antagonist, did not affect androgen stimulation of telomerase. Down-regulation by siRNA of estrogen receptor-alpha (ER alpha), but not ER beta, inhibited estrogen-stimulated telomerase function. Our results provide a mechanism for androgen therapy in bone marrow failure: androgens appear to regulate telomerase expression and activity mainly by aromatization and through ER alpha. These findings have potential implications for the choice of current androgenic compounds and the development of future agents for clinical use.

[Apoptosis induction and phosphorylated protein kinase C epsilon expression in 32D cells by sera from patients with aplastic anemia].

OBJECTIVE: To investigate the effect of phosphorylated protein kinase C epsilon (pPKC epsilon) on apoptosis of 32D cells induced by sera from patients with aplastic anemia (AA). METHODS: The expression of pPKC epsilon and apoptosis in 32D cells were measured by Western blotting and flow cytometry after incubation with sera from healthy individuals (controls, n = 8), patients with severe AA ( SAA, n = 8)and non severe AA (NSAA, n = 6). RESULTS: After incubation for 0, 12, 24, 36 and 48 hours in the presence of serum and for another 4 hours in medium deprived of serum, the levels of pPKC epsilon in cells in SAA and NSAA group increased gradually, peaked at 24 hours, and then declined (P < 0.05). Compared with that in control group (0.54 +/- 0.08), pPKC epsilon was overexpressed in both SAA group (0.90 +/- 0.10) and NSAA group (0.64 +/- 0.08) (P < 0.05) after 24 hours incubation with serum and subsequent 4 hours without serum. pPKC epsilon level was higher in SAA group than in NSAA group (P < 0.05). A greater proportion of 32D cells showed apoptosis after 24 hours incubation with sera from SAA patients [(4.05 +/- 1.05)%] and subsequent 4 hours incubation without serum than that in controls [(2.45 +/- 0.51)%, P < 0.05], which was correlated with the same serum-induced expression of pPKC epsilon (r = 0.869, P < 0.05). Although the mean level of pPKC epsilon expression was higher in NSAA group than in control group, no significant difference of apoptosis was found between the two groups [(2.45 +/- 0.51)% vs (3.24 +/- 0.56)%, P > 0.05]. CONCLUSION: Sera from both SAA and NSAA patients could upregulate the expression of pPKC epsilon in 32D cells. The SAA sera induce apoptosis in 32D cells significantly, but the latter do not.

Carbamazepine-induced hemolytic and aplastic crises associated with reduced glutathione peroxidase activity of erythrocytes.

Although pure red cell aplasia is a well-known side effect of carbamazepine treatment, intravascular hemolytic anemia is rare. We describe a 5-year-old boy who developed concurrent intravascular hemolytic anemia and erythroblastopenia, probably due to carbamazepine. Carbamazepine treatment was subsequently discontinued, and the patient was treated with red blood cell transfusions, haptoglobin, and methylprednisolone. His hematologic abnormalities were almost fully recovered within 2 weeks. Examination of the patient's and mother's erythrocyte enzyme activities revealed mildly decreased erythrocyte glutathione peroxidase (GSH-Px) activity. We speculate that patients with reduced GSH-Px activity are at a high risk of developing carbamazepine-induced hemolytic crisis and/or aplastic crisis.

Functional characterization of natural telomerase mutations found in patients with hematologic disorders.

Human telomerase hTERC RNA serves as a template for the catalytic hTERT protein to synthesize telomere repeats at chromosome ends. We have recently shown that some patients with bone marrow failure syndromes are heterozygous carriers for hTERC or hTERT mutations. These sequence variations usually lead to a compromised telomerase function by haploinsufficiency. Here, we provide functional characterization of an additional 8 distinct hTERT sequence variants and 5 hTERC variants that have recently been identified in patients with dyskeratosis congenita (DC) or aplastic anemia (AA). Among the mutations, 2 are novel telomerase variants that were identified in our cohort of patients. Whereas most of the sequence variants modulate telomerase function by haploinsufficiency, 2 hTERC variants with sequence changes located within the template region appear to act in a dominant-negative fashion. Inherited telomerase gene mutations, therefore, operate by various mechanisms to shorten telomere lengths, leading to limited marrow stem cell reserve and renewal capacity in patients with hematologic disorders.

[Effects of salidroside on bone marrow matrix metalloproteinases of bone marrow depressed anemic mice].

To examine the effect of salidroside on the expression and activities of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in bone marrow (BM) of BM depressed anemic mice by immunohistochemistry and gelatin zymography respectively, and to explore its roles in hematopoietic regulation. Immunohistochemistry showed that the expression of MMP-2 and MMP-9 of bone marrow cells (BMCs) was found in each group. Compared with control group, the expression of MMP-2 and MMP-9 was obviously increased in the model group, low-dose, middle-dose and high-dose salidroside. At day 4 after treatment of radiation and chemotherapy, the peak of the expression of MMP-2 and MMP-9 was found in middle-dose salidroside . At day 8 after treatment of radiation and chemotherapy, the peak of the expression of MMP-2 and MMP-9 was found in low-dose and middle-dose salidroside respectively. Gelatin zymography revealed that 66 kD proMMP-2, 62 kD MMP-2, 86 kD MMP-9 and 94 kD proMMP-9 were detected in control group, and the activity of MMP-9 was stronger among them. After treatment of radiation and chemotherapy, the activity of gelatinases of hemopoietic microenviroment (HM) was obviously decreased, but low-dose, middle-dose and high-dose salidroside could significantly increase the activities of proMMP-9 and MMP-9, attenuate the activity of proMMP-2. These results suggest that salidroside could promote the recovery of hematopoietic function of BM depressed anemic mice by increasing the expression and activity of MMPs, releasing the cytokines from ECM or cell membrane, repairing impaired microvessels of HM and promotion proliferation, migration and differentiation of HSCs.

Telomerase RNA levels limit the telomere length equilibrium.

Small functional RNAs play essential roles in many biological processes. Regulating the level of these small RNAs can be as important as maintaining their function in cells. The telomerase RNA is maintained in cells at a steady-state level where small changes in concentration can have a profound impact on function. Cells that have half the level of the telomerase RNA cannot maintain telomeres through many cell divisions. People who are heterozygous for telomerase RNA mutations have the diseases dyskeratosis congenita and aplastic anemia, caused by short telomeres that result in loss of tissue renewal capacity. Mice heterozygous for telomerase RNA show haploinsufficiency in telomere length maintenance and also show loss of tissue renewal capacity. It is remarkable that small changes in the level of this functional RNA can have such profound effects in cells. This tight regulation highlights the importance of controlling the action of telomerase in cells.