Mutations in EFL1, an SBDS partner, are associated with infantile pancytopenia, exocrine pancreatic insufficiency and skeletal anomalies in aShwachman-Diamond like syndrome.

BACKGROUND: For the final step of the maturation of the ribosome, the nascent 40S and 60S subunits are exported from the nucleus to the cell cytoplasm. To prevent premature association of these ribosomal subunits, eukaryotic initiation factor 6 (eIF6) binds the 60S subunit within the nucleus. Its release in the cytoplasm requires the interaction of EFL1 and SDBS proteins. In Shwachman-Diamond syndrome (SDS), a defective SDBS protein prevents eIF6 eviction, inhibiting its recycle to the nucleus and subsequent formation of the active 80S ribosome. OBJECTIVE: This study aims to identify the molecular basis of an SDS-like disease, manifested by pancytopenia, exocrine pancreatic insufficiency and skeletal abnormalities in six patients from three unrelated families. METHODS: Whole exome analysis was used for mutation identification. Fluorescence microscopy studies assessed the localisation of Tif6-GFP, the yeast eIF6 homologue, in yeast WT and mutant cells. Human and yeast EFL1 proteins, WT and mutants, were expressed in Saccharomyces cerevisiae BCY123 strain, and circular dichroism and small-angle X-ray scattering were used to assess the folding and flexibility of these proteins. Green malachite colorimetric assay was performed to determine the GTPase activity of WT and Efl1 mutants. RESULTS: Four patients were homozygous for p.R1095Q variant and two patients were homozygous for p.M882K variant in EFL1. Residue R1095 and M882 are conserved across species. Neither the GTPase activity of the mutant proteins nor its activation by the SDBD protein or the 60S ribosomal subunit were affected. Complementation of efl1Δ yeast cells with the EFL1 mutants rescued the slow growth phenotype. Nonetheless, Tif6-GFP was relocalised to the cytoplasm in mutant yeast cells in contrast to its nuclear localisation in WT cells. CONCLUSIONS: Mutations in EFL1 clinically manifest as SDS-like phenotype. Similar to the molecular pathology of SDS, mutant EFL1 proteins do not promote the release of cytoplasmic Tif6 from the 60S subunit, likely preventing the formation of mature ribosomes.

Guanine nucleotide exchange in the ribosomal GTPase EFL1 is modulated by the protein mutated in the Shwachman-Diamond syndrome.

Ribosome biogenesis in eukaryotes is a complex process that requires the participation of several accessory proteins that are not part of the mature particle. Efl1 is a yeast GTPase required for the cytoplasmic maturation of the 60S ribosomal subunit. Together with Sdo1, the yeast ortholog of the protein mutated in the Shwachman-Diamond Syndrome (SBDS), Efl1 releases the anti-association factor Tif6 from the surface of the 60S subunit allowing the assembly of mature ribosomes. We characterized the structural content and folding stability of the Saccharomyces cerevisiae and human EFL1 GTPases, as well as their enzymatic properties alone and in the presence of Sdo1 and SBDS, respectively. The human and S. cerevisiae EFL1 GTPases are composed of a mixture of α-helices and ß-sheets. Despite being orthologs, the yeast protein elicited a non-two state thermal unfolding behavior while the human EFL1 was highly resistant to thermal denaturation. Steady-state kinetic analyses indicated slow GTP hydrolysis for both EFL1 GTPases, with kcat values of 0.4 and 0.3min(-1) and Km for GTP of 110 and 180µM respectively. In the presence of the effector proteins, their kcat values remained unaltered while the Km decreased twofold suggesting that Sdo1 and SBDS act as nucleotide exchange factors.

Association of the TYMS 3G/3G genotype with poor response and GGH 354GG genotype with the bone marrow toxicity of the methotrexate in RA patients.

PURPOSE: Gamma-glutamyl hydrolase (GGH), cyclin D1 (CCND1) and thymidylate synthase (TS) genes encode enzymes that are involved in methotrexate (MTX) action. In a group of 184 RA patients treated with MTX, we have investigated whether selected polymorphisms in these genes modulate MTX efficacy and/or have impact on adverse drug effects (ADEs). METHODS: The efficacy of the MTX therapy has been estimated using the disease activity score in 28 joints (DAS28-ESR) based on EULAR criteria and relative DAS28 values (rDAS28). All adverse drug events were recorded. Patients were genotyped for selected polymorphisms of the GGH (-354 G > T and 452 C > T), CCND1 (870 A > G) and TYMS (variable number of tandem repeats, VNTR, and G to C substitution of triple repeat, 3R allele) gene. Association studies have been performed between obtained genotypes and the efficacy and toxicity of MTX. RESULTS: According to the EULAR response criteria, 146 RA patients (79.3 %) were classified as responders (good/moderate response) and 38 (20.7 %) as non-responders (poor response). Higher frequency of the TYMS 3 G/3 G genotype has been found among non-responders as compared to individuals with remaining genotypes (p = 0.02). ADEs were recorded in 53 patients. Among those patients eight experienced bone marrow toxicity, all of them carried GGH -354GG genotype (p = 0.003). No other significant association were observed. CONCLUSION: The 3 G/3 G genotype of the TYMS gene may indicate predisposition of poor response to MTX and GG genotype of GGH -354 T > G polymorphism may have high predictive value for myelosuppression in RA patients.

Role of matrix metalloproteinase-3 (MMP-3) and magnetic resonance imaging of sacroiliitis in assessing disease activity in ankylosing spondylitis.

The objective of this study is to evaluate the role of MMP-3 and MRI in assessing disease activity in sacroiliac joints of AS patients in comparison to the conventional measures Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). Serum MMP-3 was measured in 30 patients who fulfilled the modified New York criteria for AS and in ten healthy volunteers. AS patients were categorized into those having high or low MMP-3 according to a cut-off value = 7.1 ng/ml. MRI of the sacroiliac joints (SIJs) was performed on all patients. SIJs were evaluated for enhancement and subchondral bone marrow edema. Results of MMP-3 and findings on MRI were correlated with multiple clinical parameters including BASDAI, ESR and CRP. Serum MMP-3 was significantly elevated in AS patients with active disease. Elevated MMP-3 levels were significantly associated with high BASDAI (P = 0.046), but not with ESR or CRP. MRI showed bone marrow edema and enhancement of SIJs in 19/30 patients with one patient showing enhancement only. These MRI findings were not correlated with MMP-3, BASDAI, CRP or ESR. In conclusion, serum MMP-3 is an objective measure reflecting clinical disease activity in AS. Bone marrow edema and enhancement detected by MRI of SIJs is another objective measure of disease activity, but are not correlated with MMP-3 or the conventional parameters as BASDAI, ESR, or CRP. Although both MMP-3 and MRI can reflect disease activity in AS they seem to be unrelated, perhaps each is reflecting a different aspect of disease activity. MMP-3 and MRI should be considered together with BASDAI in assessing disease activity and in guiding the available recommendations for initiation of biologics in AS.

Evidence of a generalized defect of acinar cell function in Shwachman-Diamond syndrome.

BACKGROUND: : Because the acinar cells of the exocrine pancreas in patients with Shwachman-Diamond syndrome (SDS) are severely depleted, we hypothesized that a similar deficiency may be present in acinar cells of the parotid gland. PATIENTS AND METHODS: : We determined serum pancreatic isoamylase and parotid amylase activities in 16 patients with SDS, 13 healthy controls, and 13 disease controls (cystic fibrosis or fibrosing pancreatitis). Parotid amylase and electrolyte concentrations were measured in stimulated parotid gland secretions. Starch digestion was assessed by breath hydrogen testing in patients with SDS (with and without enzyme supplements) and healthy controls. RESULTS: : Serum pancreatic and parotid isoamylase values were lower in the patients with SDS than in the healthy controls (P < 0.0001 and P = 0.0002, respectively). Serum pancreatic isoamylase, but not parotid isoamylase, was significantly lower in the disease controls than in the healthy controls (P < 0.0001 and P = 0.17, respectively). Secreted parotid gland amylase concentration (units per milligram of protein) in patients with SDS was lower than that in the healthy controls (P = 0.04), whereas the disease controls were comparable to the healthy subjects (P = 0.09). Secreted parotid chloride concentration was inversely correlated with amylase concentration in the patients with SDS (P = 0.01), but no correlation was seen in the healthy controls or disease controls. When patients with SDS ingested starch without enzyme supplementation, their breath hydrogen excretion was significantly higher than that in the healthy controls (P = 0.009). Following starch ingestion with enzymes, breath hydrogen in the patients with SDS was lower (P < 0.05) than with no enzyme treatment, and no different from controls (P = 0.37). CONCLUSIONS: : Mutations in the SBDS gene cause a generalized functional abnormality of exocrine acinar cells.

Ligase IV syndrome.

Ligase IV (LIG4) syndrome belongs to the group of hereditary disorders associated with impaired DNA damage response mechanisms. Clinically and morphologically, patients affected with this syndrome are characterized by microcephaly, unusual facial features, growth retardation, developmental delay, skin anomalies and are typically pancytopenic. The disease leads to acute radiosensitivity, immunodeficiency and bone marrow abnormalities. LIG4 syndrome arises from hypomorphic mutations in the LIG4 gene encoding DNA ligase IV; a component of the nonhomologous end-joining machinery, which represents a major mechanism of repair of double strand DNA breaks in mammals. The hypomorphic mutations do not completely abolish but significantly reduce enzyme function. This results in impaired V(D)J recombination, the essential rejoining process in T- and B-cell development, in whose ligase IV plays the key role. As a consequence, patients with LIG4 syndrome frequently develop multiple immune abnormalities, clinically overlapping with severe combined immunodeficiency syndrome.

Familial leukemia.

Identification of genes responsible for rare familial cases of cancer provides genetic and biochemical insight into the mechanisms of carcinogenesis at work in the more common, sporadic occurrences of the corresponding malignancy. Hematopoietic malignancy is no exception, and considerable evidence substantiates the role of genetic factors in the risk for leukemia. In a few instances, leukemia runs in families as a single gene, Mendelian disorder. Only a few genes conferring heritable risk for leukemia are known, however, and most are responsible for bone marrow failure syndromes. Thus, the identification of additional genetic risk factors for leukemia represents both a challenge and an opportunity. The high frequency of leukemia and transient leukemia in Down syndrome is beginning to yield the secrets of its unique clinical properties. The apparent phenomenon of anticipation (a declining of age of onset with each passing generation) in familial forms of bone marrow failure and leukemia is now affirmed through its association with mutations in genes responsible for maintaining telomere length. And, for the majority of leukemia cases, as with other common diseases that are not under the influence of a single, major gene, but rather result from the additive interactions of complex genetic and environmental factors, common variants in metabolic enzymes, and other genes awaiting discovery, are being teased out.

Talc granulomatosis in the rat: the relationship between osteoblast insufficiency and adjacent bone marrow hyperplasia.

Bone loss in talc granulomatosis is paralleled by hyperplasia of bone marrow in the rat. To test the hypothetical relation between those two phenomena, bone matrix osteoinduction was employed as a model in which bone formation and bone marrow appearance are separated in time. Implantation of demineralized bone matrix to normal rats was followed by three talc injections (one weekly), starting 1 week after matrix implantation. Implants of demineralized bone and [3H]thymidine-labeled tibial metaphyses from talc-injected and normal rats were analyzed histologically and evaluated for alkaline and tartrate-resistant acid phosphatase activity on days 7, 14, 21, and 30 after matrix implantation. Analysis of tibial autoradiographs showed a marked growth arrest and bone marrow hyperplasia in talc-injected rats 7 days after first talc injection. Alkaline phosphatase activity in homogenates of bone implants was low in talc-injected rats on day 14 after implantation. Moreover, histology of the bone implants showed numerical and functional inhibition of osteoblasts on the same day, causing marked growth delay. Bone marrow appeared as late as day 21 after bone matrix implantation. We conclude that hyperplasia of the adjacent bone marrow is not the cause of bone loss in talc granulomatosis, but rather its compensatory consequence.

Detection of tyrosine hydroxylase mRNA and minimal neuroblastoma cells by the reverse transcription-polymerase chain reaction.

To facilitate the diagnosis of bone marrow metastasis in neuroblastoma, we have developed a method of amplifying and detecting the tyrosine hydroxylase (TH) mRNA sequence in bone marrow cells using a combination of reverse transcription and the polymerase chain reaction (RT/PCR). By this method, the sequence of TH was detected clearly in the neuroblastoma tissues of all 6 patients and not detected in the bone marrow cells of any of the 9 negative control children. In a reconstitution experiment, 1 neuroblastoma cell per 100,000 normal bone marrow cells could be detected, thus indicating the great sensitivity of this method. Based on these results, this technique may be of value in the diagnosis and treatment follow-up of bone marrow metastasis of neuroblastoma.

Genetic differences in susceptibility to chemically induced myelotoxicity and leukemia.

The Ah locus represents a complex "cluster" of genese controlling the induction of numerous drug-metabolizing enzyme "activities" by polycyclic aromatic compounds. Allelic differences at the Ah locus are reflected in the large differences in inducibility of cytochrome P1-450 and benzo[a]pyrene metabolism in numerous tissues when the mice receive the chemical daily in their diet. This experimental model system offers to the hematologist and clinical pharmacologist a means to study genetic differences in toxic chemical depression of the bone marrow, as well as a potential model to study aplastic anemia and leukemia explainable on a single-gene basis. The genetically "responsive" individual who is at increased risk for cancer caused by subcutaneous or topical or intratracheal polycyclic hydrocarbons is at decreased risk for toxicity of the bone marrow and leukemia caused by oral benzo[a]pyrene (when compared with the genetically "nonresponsive" individual receiving the same dose of the same xenobiotic). In other words, tissue sites in direct contact with the carcinogen develop cancer in responsive animals because of induced P1-450; tissues in distant sites of the body may develop malignancy in nonresponsive animals because more carcinogen reaches that tissue due to decreased P1-450 induction all over the body and therefore decreased detoxication. Not only the dct with the carcinogen develop cancer in responsive animals because of induced P1-450; tissues in distant sites of the body may develop malignancy in nonresponsive animals because more carcinogen reaches that tissue due to decreased P1-450 induction all over the body and therefore decreased detoxication. Not only the dct with the carcinogen develop cancer in responsive animals because of induced P1-450; tissues in distant sites of the body may develop malignancy in nonresponsive animals because more carcinogen reaches that tissue due to decreased P1-450 induction all over the body and therefore decreased detoxication. Not only the dose but the route of administration and the tissue in which the malignancy or toxicity develops are therefore very important in the interpretation of data from tumorigenesis or toxicity experiments involving P1-450 inducers such as polycyclic hydrocarbons. There exists sufficient evidence that heritable variation of the Ah locus occurs in man. Growing evidence indicates that persons with higher aryl hydrocarbon hydroxylase inducibility in their cultured mitogen-activated lymphocytes may have a statistically significantly increased risk for certain types of cancer and drug toxicity. It remains to be determined at the present time, however, whether this genotype can be used as a biochemical marker in the individual patient for predicting increased susceptibility to certain types of environmentally caused cancers or toxicity in man.

Glutathione S-transferase in bone marrow metastases of disseminated neuroblastoma.

Antisera to each of the three main cytosolic forms of glutathione S-transferase (GST; alpha, mu, and pi) has been used to characterise GST expression by metastatic neuroblastoma in bone marrow trephine biopsies taken from 15 patients at presentation and from five of this group at relapse. There was no correlation between expression of extra-nuclear alpha or mu GST and outcome, and no consistent pattern at relapse. Seven of eight expressing nuclear pi GST at presentation died of resistant disease. Three of five cases with no detectable nuclear pi class GST remain alive and disease free. The results provide no encouragement for further investigation of alpha or mu GST in this disease but larger studies of uniformly treated patients may show whether nuclear pi GST expression at presentation indicates likely relapse.

Mechanisms of the acquired erythrocyte enzyme deficiencies in blood diseases.

Acquired enzymatic activity defects of erythrocyte pyruvate kinase, glucose phosphate isomerase and phosphofructokinase have been studied in patients with acute myeloid leukemias, sideroblastic refractory anemias and unclassified acquired dyserythropoiesis. 6 patients with acute myeloid leukemia had a lowered erythrocyte pyruvate kinase activity; in 5 of them the concentration of the "pyruvate kinase"-antigen was parallely decreased, in such a manner that the ratio enzyme activity/immunologic reactivity (i.e. the molecular specific activity) was normal. In 1 patient with acute leukemia, 4 with refractory anemia and 1 with acquired dyserythropoiesis the defect of the pyruvate kinase activity was associated with a normal antigen concentration (and, therefore, the molecular specific activity in whole hemolysate was lowered). The enzyme activity was restored by incubation with SH reagents in two cases and by partial purification as often as it was performed. The electrofocusing pattern of erythrocyte pyruvate kinase was normal in both these types of defects. In two patients with so-called "acquired dyserythropoiesis" an erythrocyte glucose phosphate isomerase deficiency has been detected; in both the cases it was associated with a parallel decrease of the antigen concentration. The residual enzyme had a normal electrofocusing and electrophoretic pattern and a normal heat stability; the enzyme activity could not be restored by any treatment. In 1 patient with erythroleukemia and in 1 other with acquired dyserythropoiesis the erythrocyte phosphofructokinase activity was lowered. The enzyme activity was not restored by cross incubation in isologous plasma or by the SH reagents. In one case immunologic study could be performed, indicating that the enzyme defect was mainly due to the decreased ratio of the muscle type subunit of the erythrocyte phosphofructokinase. The electrofocusing pattern of deficient phosphofructokinases was normal. Finally, we point out the probable existence of several direct mechanisms, genetic and post translational, accounting for the acquired enzyme defects of red blood cells in various blood disorders.

[Leukemization of hematosarcomas with primary involvement of the skin]. / Leikemizatsiia gematosarkom s pervichnym porazheniem kozhi.

Clinicomorphological investigation of 81 patients with different types of skin hematosarcomas was conducted. Stage IV of disease was diagnosed in 53 patients, leukemic involvement developed in 18 of them (34%). Leukemic bone involvement was shown to be the main cause of death in myelosarcomas and lymphoblastic lymphomas. There were no significant differences in the frequency of leukemic bone marrow involvement with relation to the site and nature of primary skin involvement. A clinical course of disease was determined by a skin tumor histological type. Some morphological features of bone marrow lymphoblasts in leukemic bone marrow involvement in skin hematosarcomas (irregular cell shape, a moderate nuclear-cytoplasmic ratio, azurophilic granulation, fine-grained patterns of PAS-positive substance) distinguished them from lymphoblasts in leukemic bone marrow involvement in hematosarcomas with a primary focus of another site.

Pluripotent stem cell models of Shwachman-Diamond syndrome reveal a common mechanism for pancreatic and hematopoietic dysfunction.

Shwachman-Diamond syndrome (SDS), a rare autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and hematopoietic dysfunction, is caused by mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. We created human pluripotent stem cell models of SDS through knockdown of SBDS in human embryonic stem cells (hESCs) and generation of induced pluripotent stem cell (iPSC) lines from two patients with SDS. SBDS-deficient hESCs and iPSCs manifest deficits in exocrine pancreatic and hematopoietic differentiation in vitro, enhanced apoptosis, and elevated protease levels in culture supernatants, which could be reversed by restoring SBDS protein expression through transgene rescue or by supplementing culture media with protease inhibitors. Protease-mediated autodigestion provides a mechanistic link between the pancreatic and hematopoietic phenotypes in SDS, highlighting the utility of hESCs and iPSCs in obtaining novel insights into human disease.

TAK1 is required for the survival of hematopoietic cells and hepatocytes in mice.

Transforming growth factor beta-activated kinase 1 (TAK1), a member of the MAPKKK family, is a key mediator of proinflammatory and stress signals. Activation of TAK1 by proinflammatory cytokines and T and B cell receptors induces the nuclear localization of nuclear factor kappaB (NF-kappaB) and the activation of c-Jun N-terminal kinase (JNK)/AP1 and P38, which play important roles in mediating inflammation, immune responses, T and B cell activation, and epithelial cell survival. Here, we report that TAK1 is critical for the survival of both hematopoietic cells and hepatocytes. Deletion of TAK1 results in bone marrow (BM) and liver failure in mice due to the massive apoptotic death of hematopoietic cells and hepatocytes. Hematopoietic stem cells and progenitors were among those hematopoietic cells affected by TAK1 deletion-induced cell death. This apoptotic cell death is autonomous, as demonstrated by reciprocal BM transplantation. Deletion of TAK1 resulted in the inactivation of both JNK and NF-kappaB signaling, as well as the down-regulation of expression of prosurvival genes.

Intra-ethnic differences in genetic variants of the UGT-glucuronosyltransferase 1A1 gene in Chinese populations.

Variants within the human UGT1A1 gene are associated with irinotecan induced severely adverse reactions and hyperbilirubinemia. Intra-ethnic differences in the genetic variation and haplotypes of UGT1A1 gene have been analyzed in the present study. Relationship between the concentrations of total serum bilirubin (T-bil) and haplotype structure of UGT1A1 in healthy people were also evaluated. We genotyped five functional polymorphisms including -3279T>G and -3156G>A in the enhancer region, (TA)6>7 in the TATA box, and 211G>A (G71R), 686C>A (P229Q) in the exon1 region of UGT1A1 in three groups of healthy Chinese ethnic populations, consisting of 264 subjects of She origin, 539 of Han origin and 273 of Dong origin. The distribution of -3279T>G, (TA)6>7, 211G>A of UGT1A1 differed greatly as between the three ethnic groups. All of six haplotypes differed considerably between at least two of the three groups, which highlighted the need to analyze clinically irinotecan toxicity relevant SNPs and haplotypes in a variety of different racial groups within the Chinese population. Total bilirubin concentration in homozygous carriers of the -3279G and (TA)7 allele were significantly higher than those in heterozygous carriers or homozygous carriers of wild-type alleles. Carriers of the variant haplotypes (-3279G; -3156A; (TA)7; 211G; 686C) had higher serum T-Bil concentrations compared with the other groups. Our results indicate that heterogeneity among different ethnic populations is possibly the result of microevolution and is relevant to studies into the effect of tailored drug treatment.