Academic literature on the topic 'Mutation dw'

The dwarf (dw/dw) rat differs from all other rodent models of GH deficiency in that its pituitary prolactin (PRL) content is normal or even increased. We have now studied this throughout postnatal development, using a combination of immunocytochemistry, RIA and fluorescence-activated cell sorting (FACS) and analysis. Compared with normal Albino Swiss (AS) rats, adult dw/dw rats showed a profound reduction in pituitary GH content accompanied by increased PRL content, significantly so in females (AS vs dw/dw; P<0.01). Somatotroph hypoplasia was evident in the adult dw/dw rats, with most GH(+ve) cells showing weak immunostaining, whereas many more strongly stained PRL cells were evident in pituitary sections from dw/dw rats. Facs analysis confirmed both somatotroph hypoplasia and relative lactotroph hyperplasia in dw/dw rats at all ages studied (9-144 days); the difference in somatotrophs increased with age whereas the difference in lactotrophs declined with age. At 9 days, the percentage of lactotrophs was 10-fold higher in dw/dw rats than in AS rats. Young dw/dw rats also had a higher proportion of mammosomatotrophs than AS rats, although this difference disappeared as the mammosomatotroph proportions increased with age in both strains. GHRH released GH from both dw/dw and as cells maintained in culture for 5 days. The sensitivity to GHRH and the amount of GH released was lower in the dw/dw cultures, mostly explained by their fewer GH cells and lower initial GH content. GHRH increased cAMP in as but not in dw/dw cultures, even when these were greatly enriched for dw/dw somatotrophs by FACS sorting prior to culture. These results suggest that GHRH-induced cAMP stimulation is required for trophic effects on GH synthesis and somatotroph proliferation, but is not required for GHRH-stimulated GH release. The inverse changes in somatotroph and lactotroph numbers suggest that the dw/dw mutation disturbs the mechanism that specifies and retains appropriate numbers of somatotrophs in their differentiated state, and results in a higher proportion of the remaining cells progressing to lactotrophs. The dw/dw phenotype is thus not confined to somatotrophs.

Presoaked (24 h at 20 °C) seeds of Cuphea tolucana Peyr. and C. wrightii A. Gray were treated with mutagens at 20 °C in two experiments. Experiment 1 treatments were: distilled water (DW), 0.05 M PO4 buffer (pH 7), 0.01 M ethyl methanesulfonate (EMS) (each applied for 8 h), 0.02 M EMS (applied for 4 h), 0.04 EMS M, 0.08 M EMS, or 0.16 M EMS (each applied for 2 h). Experiment 2 treatments were: DW, 0.1 M PO4 buffer (pH 3), 0.0005 M sodium azide (SA), 0.001 M SA, or 0.002 M SA (each applied for 2 h). None of the treatments had significant effects on emergence and height of M1 plants nor were any macro-mutations noted in the M2 generations. In a third experiment, DW, 0.04 M EMS, or 0.001 M SA were applied for 2 h at 30 °C to presoaked (48 or 72 h at 30 °C) seeds of C. tolucana and C. wrightii. Compared to EMS, SA had deleterious effects on height in the M1, emergence was better for C. tolucana than for C. wrightii, and C. wrightii plants grew taller after a 72-h pre-soak than after a 48-h presoak. M2 progenies were evaluated in the field. None of the presoak-treatment combinations increased variation significantly in several quantitative characters, no macro-mutations were detected in C. wrightii, but a fertile, small-leaved, decumbent mutant with very short internodes was noted in C. tolucana. Mutation rates were greatest for the 72 h presoak-EMS combination.Key words: Cuphea tolucana, Cuphea wrightii, ethyl methanesulfonate, sodium azide, medium-chain triglycerides, lauric acid

Abstract To gain insight into the pathways by which caloric restriction (CR) slows aging, gene expression levels were assessed for each of 2352 genes in the livers of 9-month-old CR and control mice. A total of 352 genes were found to be significantly increased or decreased by CR. The distribution of affected genes among functional classes was similar to the distribution of genes within the test set. Surprisingly, a disruption or knockout of the gene for the GH receptor (GHR-KO), which also produces life extension, had a much smaller effect on gene expression, with no more than 10 genes meeting the selection criterion. There was, however, an interaction between the GHR-KO mutation and the CR diet: the effects of CR on gene expression were significantly lower in GHR-KO mice than in control mice. Of the 352 genes altered significantly by CR, 29 had shown a significant and parallel alteration in expression in a previous study of liver gene expression that compared mice of the long-lived Snell dwarf stock (dw/dw) to controls. These 29 genes, altered both by CR and in dwarf mice, provide a list of biochemical features common to both models of delayed aging, and thus merit confirmation and more detailed study.

Abstract Liver iron concentration (LIC) is a known and accurate marker of iron accumulation and is widely utilized to monitor iron chelation therapy in multiply transfused patients with Beta thalassemia major. Iron concentration in the bone marrow has not been studied and reported before. We utilized atomic absorption spectrophotometry to measure the iron content of marrow biopsy (BIC) in 102 thalassemia patients in various phase of treatment, 74 of them during the course of the disease and 28 patients after successful allogeneic marrow transplant. We observed BIC values below 0.5 mg/g dry weight in 7 healthy donors used as controls. Mean and median BIC were 4.67 and 2.70 (range 0.05 – 59.9) mg/g dw, in 101 valuable patients. Bone Marrow iron concentration (BIC) was calculated at the same time of LIC for each patient and a ratio LIC/BIC was generated. LIC/BIC ratio below 3, ratio between 3 and 10, and above 10 identified three categories of patients each with significant linear correlation between LIC and BIC (0.74; 0.76; 0.81 respectively). Twenty eight patients were in the first category, 48 in the second, and 25 in the third. Mean BIC was 9.47, 3.7, 1.2 mg/g dw and mean LIC was 12.7, 19.6, 22.3 mg/g dw respectively for CAT1, CAT2 and CAT3. Patients were also classified in class of risk for transplant, based on hepatomegaly, presence of liver fibrosis and history of regular or irregular iron chelation. BIC was higher in class 3 patients and in the irregularly chelated patients. Patients in class 1 were prevalently in CAT1 or 2, patients in class 3 were prevalently in CAT2 or 3. BIC value in each of the 3 categories correlated significantly with the whole body iron (WBI) amount, and WBI was significantly different in the three categories being lower in CAT1, that have higher BIC ( 3927 mg; 5894 mg, 7030 mg in CAT1, 2 and 3; p 0.01). Iron chelation quality (regular vs irregular) correlated with BIC value and with BIC category, majority of regularly chelated patients were in the Category 1 vs Category 2 or 3 (p 0.01). Patients before transplant were prevalently in CAT1 and 2, while those post transplant were mostly in CAT3, twelve patients that were studied both before and after transplant, changed from cat 1 to 2 or from cat 2 to 3. Their BIC changed significantly decreasing after BMT (median BIC was 7.8 before transplant and 2.25 after, p=0.002) To investigate the role of genetic hemochromatosis mutations, H63D region and Hamp region in 63 patients were also studied. Mean BIC was 3.08 in 16 patients with H63D mutation compared to 5.59 in those without mutation, and 11/16 had BIC below the median, p=0.03. Significantly more patients with H63D mutation were in CAT3 (p 0.02). Apparently H63D mutation favours accumulation of iron in the body, that was higher in CAT3, but participate also to lowering utilization of introduced iron. In conclusion, patients in category 1 had lower LIC and higher BIC, comprehended 50% of the patients in class 1 of risk, and 45% of the regularly chelated patients. On the contrary, majority of patients post transplant independently of having or not performed an iron removal program were in CAT3. We can draw conclusion that BIC related categories, as described here, give information on the balance between accumulation and utilization of iron. Patients belonging to BIC-Cat 1 correspond to patients that have been treated adequately and with lower iron body burden, better chelation history. They have higher BIC value and lower LIC value. They also are in pre-transplant phase, with active beta-thalassemia, when ineffective erythropoiesis and marrow expansion are more prominent. Further studies will be necessary to confirm the association of marrow iron content with the marrow functionality and expansion.

GH levels increase to high concentrations immediately before puberty then progressively decline with age. GH deficiency (GHD) originating in childhood is treated with GH supplementation to foster somatic development during adolescence. It is not clear if or how early GH replacement affects memory in adulthood, or whether it can prevent the cognitive deficits commonly observed in adults with childhood-onset GHD. Rats homozygous for the Dw-4 mutation (dwarf) do not exhibit the normal increase in GH at 4 weeks of age when GH levels normally rise and are used to model childhood or early-onset GHD (EOGHD). One group of these rats was injected with GH from 4 to 14 weeks of age to model GH supplementation during adolescence with GHD beginning in adulthood (adult-onset GHD; AOGHD). Another group received GH from 4 weeks throughout the lifespan to model normal lifespan GH (GH-replete). Age-matched, Dw-4 heterozygous rats (HZ) do not express the dwarf phenotype and were used as controls. At 8 and 18 months of age, spatial learning in the water maze was assessed. At 8 months of age all experimental groups were equally proficient. However, at 18 months of age, the EOGHD group had poor spatial learning compared to the AOGHD, GH-replete, and HZ groups. Our data indicate that GHD during adolescence has negative effects on learning and memory that emerge by middle-age unless prevented by GH supplementation.

Abstract Clinical data: Subject: Male, 45 years old, diagnosed in 2001 with Ph+ CMLCP (low Sokalrisk).From 2001 to 2005 he was treated with Hydrea and Interferon with low dose Cytarabine. In 2005 he was switched to imatinib 400 mg/day. Due to cytogenetic resistance Imatinib dose was escalated to 600mg/day in 2006 and 800mg/day in 2007. No cytogenetic response was obtained. In 2009 patient was switched to nilotinib. Nilotinib therapy was discontinued in 2012 due to loss of complete hematologic response. Patient refuses unrelated donor allo HSCT. INFa 3000 U/3 times a day was given with prolonged stable complete hematologic response. Myeloid blasts crisis was diagnosed in Jan 2013. (The time patient was hospitalized to Almazov medical research center). Dasatinib 140mg/day was started without rapid blast reduction.The response lost in a few weeks, followed by 2 couses of FLAG regimen. Patient died in Dec 2013. BCR-ABL KD mutations history: 2007- no mutations found; 2009- no mutations found, February 2013 -L248V mutation; July 2013 mutation analysis returns no result due to the poor sequence quality;T315I mutation revealed in samples obtained from October 2013. BCR-ABL mutation analysis: Bcr-Abl analysis performed as described before (Branford et al., 2002). KD sequence was amplified from cDNA using seminested PCR, PCR product extracted from gel and sequenced in the both directions on ABI 3130 genetic analyzer. For confirmation we used genomic DNA extracted from stored peripheral blood samples. Primers specifically amplifying exon 4 and 5 of ABL used for genomic DNA analysis. Analysis of the sequences was done using UGENE software. Results: We analyzed two sets of probes- one dated March 2011 and another dated October 2013. After performing semi-nested PCR on BCR-ABL KD sequence we found at least two bands in each of the samples. Sequencing of this bands revealed 81 bp deletion translated into absence of 27 aa position at 248-274. Genomic DNA analysis confirmed alternative splicing and appearance of the truncated form. We found SNP resulted in T315I mutation presented either in full or in short isoform of BCR-ABL in samples from October 2013. Also we confirmed L248V mutation presented in all analyzed samples. Conclusion: SNP that cause L248V mutation in BCR-ABL transcript known to activate cryptic promotor in ABL exon4 with subsequent appearance of alternative splicing form described before (Gruber et al., 2006). Truncated form known to be kinase dead and have no influence on disease course (Shebenou et al., 2008). On the other hand it disturbs the Sanger sequence quality, masking the appearance of the additional mutations. Extensive attention requires in obtain proper sequence quality: use genomic DNA for Sanger sequencing or alternative sequencing methods, e.g. single-molecule long-read sequencing for revealing mutations in multiple splice isoforms. References: Branford S, Rudzki Z, Walsh S, Grigg A, Arthur C, Taylor K, Herrmann R, Lynch KP, Hughes TP. High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571)resistance. Blood. 2002 May 1;99(9):3472-5. Gruber FX, Hjorth-Hansen H, Mikkola I, Stenke L, Johansen T. A novel Bcr-Abl splice isoform is associated with the L248V mutation in CML patients with acquired resistance to imatinib. Leukemia. 2006 Nov;20(11):2057-60. Sherbenou DW, Hantschel O, Turaga L, Kaupe I, Willis S, Bumm T, Press RD,Superti-Furga G, Druker BJ, Deininger MW. Characterization of BCR-ABL deletion mutants from patients with chronic myeloid leukemia.Leukemia. 2008, Jun;22(6):1184-90. Disclosures Lomaya: Novartis: Consultancy. Zaritskey:University of Heidelberg: Research Funding; Novartis: Consultancy.

Abstract Introduction: BCR-ABL kinase domain (KD) mutations have well established role in tyrosine kinase inhibitors (TKIs) resistance and disease progression in chronic myeloid leukemia (CML)1. In recent years, compound BCR-ABL mutations have emerged as a new threat to CML patients by causing higher degrees of resistance involving multiple TKIs, including ponatinib2. However, there are limited reports about association of compound BCR-ABL mutations with disease progression in imatinib sensitive CML patients3. Furthermore, BCR-ABL mutation detection is currently recommended only in case of drug resistance and disease progression and clinical significance of BCR-ABL mutation detection in TKI responder chronic phase CML is not well documented4. Therefore, we investigated presence of ABL-KD mutations in chronic phase (CP) and advanced phase imatinib sensitive CML to find out association of BCR-ABL mutations with progression to advanced disease phases in CML patients. . Patients and Methods: Imatinib sensitive CML patients in CP and advanced phases of the disease were included in the study.All CP patients were incomplete hematological, complete cytogenetic and major molecular responses. Due to specific study objectives, patients manifesting drug resistance during follow-up studies were excluded from the study. A total of 90 imatinib sensitive CML patients (CP=41, late CP=33, and accelerated phase=16) were finally available for analysis. All patients as well as 10 healthy controls were investigated for BCR-ABL mutations using Sanger sequencing. All response criteria were per European LeukemiaNet guidelines4. Data was analyzed using SPSS software (version 19). Results: Mean age of the patients was 33 years (Table 1). Eleven out of 33 (33.3%) patients in late-CP CML harbored 24 types of point mutations, out of which eight (72.72%) harbored compound mutated sites (Figure 1, Table 2). E355G (3.33%) was the most prevalent mutant. Five patients (45.45%), all of which had compound mutations, progressed to advanced phases of disease during follow up studies. No BCR-ABL mutation was detected in healthy subjects and in early CP CML patients. Therefore, early CP CML patients served as additional control in this study. BCR-ABL mutations were found in 3 accelerated phase patients as well. Late-CP mutations were associated with elevated platelet count (p= 0.037) and male gender (p= 0.049). The median overall survival and event free survival of CML patients (n=90) was 6.98 and 5.8 years respectively. Seven year survival was found to be 94.2%. Discussion and conclusions: Compound BCR-ABL mutations were associated with progression to advanced disease in imatinib sensitive late-CP CML patients. Although single BCR-ABL mutations have previously been found to cause CML progression5, this is first report of association of compound BCR-ABL mutations with disease progression in stable imatinib responders at late CP. Detection of new mutations can help in defining mechanism of CML progression6. Moreover, BCR-ABL mutation detection in late CP CML patients sensitive to TKI treatment can help in early assessment of risk for disease progression and/or drug resistance and subsequent clinical intervention to delay disease progression which is a major challenge of CML therapy in TKI era7. References: 1. Haznedaroglu IC. Mediterranean journal of hematology and infectious diseases 2015;7. 2. Khorashad JS, Kelley TW, Szankasi P, Mason CC, Soverini S, Adrian LT, Eide CA, Zabriskie MS, Lange T, Estrada JC. Blood 2013;121:489-98. 3. Deininger MW, Hodgson JG, Shah NP, Cortes JE, Kim DW, Nicolini FE, Talpaz M,Baccarani M, Müller MC, Li J, Parker WT, Lustgarten S, Clackson T, Haluska FG,Guilhot F, Kantarjian HM, Soverini S, Hochhaus A, Hughes TP, Rivera VM, Branford S. Blood. 2016 Feb 11;127(6):703-12.dddd 4. 7. Baccarani M, Deininger MW, Rosti G, Hochhaus A, Soverini S, Apperley JF, Cervantes F, Clark RE, Cortes JE, Guilhot F. Blood 2013;122:872-84. 5. Carella AM, Garuti A, Cirmena G, Catania G, Rocco I, Palermo C, Pica G, Pierri I, Miglino M, Ballestrero A, Gobbi M, Patrone F. Leukemia & lymphoma 2010;51:275-8. 6. Giotopoulos G, van der Weyden L, Osaki H, Rust AG, Gallipoli P, Meduri E,Horton SJ, Chan WI, Foster D, Prinjha RK, Pimanda JE, Tenen DG, Vassiliou GS,Koschmieder S, Adams DJ, Huntly BJ. J Exp Med. 2015 Sep 21;212(10):1551-69. 7. Mukherjee S, Kalaycio M. Curr Hematol Malig Rep. 2016 Apr;11(2):86-93. Disclosures No relevant conflicts of interest to declare.

Abstract Factor XI (FXI) is a 160kD homodimeric component of the intrinsic pathway of the blood coagulation system. The FXI Apple 4 domain (A4) mediates homodimer formation even when the cysteine responsible for the intermolecular disulfide is mutated to serine (C321S). Preliminary NMR structural studies of the isolated wild type (wt) A4 domain showed a flattened, planar structure (Samuel D, Cheng H, Riley PW, Walsh PN, Roder H. Blood104, 481a, 2004), compared to a more globular structure consisting of two layers of β-sheet and a flanking α-helix that was observed on further analysis of wt and C321S A4. Hydrogen exchange NMR and surface NMR analysis (SEAHSQC), as well as temperature dependent hydrogen exchange NMR showed that in C321S and wt A4, the loop surrounding C321 or S321 is flexible and exposed to the solvent. Thus, the globular structure is the major species in both wt and C321S A4, but the planar structure is present as well. Fluorescence anisotropy studies of C321S A4 yielded a Kd value of ~64 nM (monomeric protein concentration), compared with ~72 nM for a dissociable mutant full-length FXI (G326C) obtained by analytical ultracentrifugation (Sinha D, Marcinkiewicz M, Lear JD, Walsh PN. Biochemistry, in press, 2005), suggesting that only the A4 domain mediates dimer formation in the intact protein. In addition, fluorescence and circular dichroism (CD) studies of the folding equilibrium of C321S A4 indicate that starting from the unfolded monomeric state, partial folding precedes dimerization. This folding mechanism for FXI A4 is consistent with the observed failure of a naturally occurring mutant (FXI F283L) to dimerize intracellularly (Meijers JC, Davie EW, Chung DW. Blood79, 1435–1440, 1992). To determine the structural consequences of the mutation, F283L was introduced into the noncovalent dimer C321S A4, which increased the Kd of the homodimer interaction to ~5 μM, but did not change the secondary structure (from comparison of the CD spectra), or the high degree of thermostability observed for C321S A4. Since the F283 sidechain has been tentatively assigned to the surface of FXI A4 near the interface, the mutation to Leu may significantly alter the interface, thereby raising the Kd but not changing the overall secondary structure significantly. In summary, there appears to be a mechanism by which a conformational change or productive folding process occurs in the monomer state that activates it for native dimer formation. Also, inserting the F283L (type III) patient mutation does affect the dimer stability, but not the overall secondary structure.