Heterozygous RTEL1 Variants Are Associated with Bone Marrow Failure and Abnormal Clinical Phenotype

Abstract Introduction Heterozygous RTEL1 mutations have recently been described in familial pulmonary fibrosis (PF) but are not known to be associated with cytopenias or bone marrow failure (BMF), in contrast to heterozygous mutations in other telomere maintenance genes TERT, TERC and TINF2. Constitutional BMF syndromes typically present with less severe pancytopenia and it is often unclear if they have hypocellular MDS (hypoMDS) or non-severe AA (NSAA) morphologically. Methods We screened 284 patients with idiopathic AA or uncharacterised BMF and 172 patients with MDS or acute myeloid leukemia (AML) for TL and RTEL1 variants, and for the other currently known telomere gene complex (TGC) mutations, after excluding patients with Fanconi anemia, DBA or other known inherited BMF syndrome. TL was measured using a monochrome multiplex quantitative PCR method on peripheral blood mononuclear cells. Illumina Nextera-amplicon sequencing was used to screen exons of the DC genes (DKC1, TERC, TERT, RTEL1, CTC1, NHP10, NOP2, USB1, WRAP53, TINF2, PARN and ACD) by MiSeq platform. Constitutional DNA was also analysed in 10 patients (skin 9, buccal swab 1) with RTEL1 variants. A targeted gene panel of 24 genes of an Illumina Tru-Seq Custom Amplicon workflow and platform was used to identify genes frequently mutated in MDS/AML. Impact of mutations was predicted based on 3D structure information from comparative modelling for the helicase domain, comprising the HD1 and HD2 subdomains, a Fe-S cluster and an ARCH domain, and for two harmonin-like (HML) domains and a RING finger domain, located in the C-terminal regulatory region of RTEL1. Results Heterozygous RTEL1 variants were identified in 20 (4.4%) patients. RTEL1 variant allele frequency (VAF) was 45-70% consistent with heterozygous inheritance in all cases. TL was short in 18 (90%) patients, being < 1st centile in 15 and <10th centile in 3. 2 patients had normal TL, <20th centile and >50th centile, respectively. Median age was 35 years (range 18-73). 15/20 (75%) had a hypocellular BM (7 hypoMDS, 5 non-severe AA, 3 ICUS), and 1 each with RAEB1, RAEB2, CMML1, AML and isolated macrocytosis. 3 patients had abnormal karyotype: +8 (hypoMDS), -Y,+1,del(1) (hypoMDS), del7q (RAEB1). 2 other patients with hypoMDS had somatic mutations: U2AF1 (30% VAF) with ASXL1 (27% VAF); U2AF1 (43% VAF). Lung abnormalities were early PF (1), interstitial lung disease (1), and abnormal lung function with reduced TLCO (1) and an obstructive picture (1). Liver fibrosis with portal hypertension and varices and reticulate skin pigmentation were present in the patient with ILD, 2 patients had dystrophic nails, and 1 unexplained mild hepato-splenomegaly. 2 patients had familial MDS, 5 had a family history of cancers affecting first-degree relatives, and 2 had skeletal and cartilage anomalies, associated with learning difficulties in 1 patient. 8/15 patients with hypocellular BM required no treatment (5 hypoMDS and 3 NSAA), one hypoMDS had CR with ciclosporin and another underwent successful unrelated donor stem cell transplant; for NSAA, 2 received ATG with CSA, with PR followed by relapse in one, the other was lost to follow up, and 1 was androgen responsive. 16/20 (80%) patients are alive; 3 patients with RAEB or AML died of progressive disease and 1 patient with ICUS and severe constitutional features died from lymphoma 10 years after presentation. Mutations were spread throughout the entire RTEL1 sequence (summarised in Figure). 3D structure analysis predicted the missense RTEL1 mutations would result in disturbance of the FeS cluster and/or interfere with DNA binding, destabilisation of the HD1, HD2 or the ARCH sub-domains of the helicase domain, or destabilisation of inter-domain interactions. One HML1 mutation occurred in a loop opposite the putative ligand binding site and the rest in the variable regions outside the conserved domains. RTEL1 variants were associated with TERT mutations in 4 patients, of which 3 were known pathogenetic and 1 novel TERT mutation with low telomerase activity on TRAP assay confirming its pathogenetic nature. Conclusions We show for the first time that heterozygous RTEL1 mutations occur in 4.4% of patients, most commonly in young patients with a hypocellular BM, and often a family history of BMF/malignancy, and less often with high risk MDS/AML. Abnormal clinical features were present in a third of patients, some similar to but others distinct from dyskeratosis congenita. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Comprehensive Identification Of Germline Alterations In Telomerase Complex Genes By Whole Exome Sequencing Of MDS and Related Myeloid Neoplasms

Blood ◽

10.1182/blood.v122.21.522.522 ◽

2013 ◽

Vol 122 (21) ◽

pp. 522-522

Author(s):

Swapna Thota ◽

Sarah McMahon ◽

Bartlomiej Przychodzen ◽

Thomas LaFramboise ◽

Hideki Makishima ◽

...

Keyword(s):

Bone Marrow ◽

Family History ◽

Research Funding ◽

Somatic Mutations ◽

Germ Line ◽

Bone Marrow Failure ◽

Telomere Maintenance ◽

Myeloid Neoplasms ◽

History Of ◽

The Mean

Abstract In addition to classical familiar forms of bone marrow failure, some cases of aplastic anemia (AA) have been linked to inherited germ line polymorphism/mutations of telomerase machinery, leading to excessive telomere shortening. Germline telomere maintenance machinery mutations have been also been found in a proportion of acute myeloid leukemia (AML) and Myelodysplastic syndromes (MDS) patients (pts). However, the molecular pathogenesis of adult MDS and AML is complex and determination of genetic risk factors in addition to established familial and congenital syndromes has been difficult. To date targeted sequencing has been used for mutational screens with the inherent limitations of limited exome coverage, empiric bias and labor intensity. New generation (NGS) whole genome approaches prioritize somatic mutations as initial discovery targets, but the availability of sequenced cohorts allows also for detection of germline lesions both in a targeted and an unbiased fashion. Using NGS we studied 136 pts (mean age, 68.8 years, range 41-85) with MDS and related myeloid neoplasms for the presence of non-synonymous polymorphisms (SNV), which could affect telomerase machinery. These genes included TERT, DKC1, SMG6, NOP10, POT1, WRAP53, NHP2, GAR1, TINF2. No somatic defects of the telomerase complex were detected. We focused on novel sequence alterations or those described in available databases with a population allelic frequency of less than 5%. We identified 45 non-synonymous germline sequence alterations in 39 cases (32%). Most frequent SNV were found in TERT (n=15), DKC1 (n=7), SMG6 (n=6), NOP10 (n=4), POT1 (n=4), WRAP53 (n=4), while observations of NHP2 (n=3), GAR1 (n=1), TINF2 (n=1) were less prevalent. These variants were distributed in an almost mutually exclusive manner. Out of 3 variants in TERT, p.H412Y (n=3) and p.A279T (n=9) were reported to be pathogenic in bone marrow failure syndromes. In addition, p.A999T found in 8 cases in our cohort could also be pathogenic since it is less frequent in healthy controls. Similarly, p.441_442del (n=1), located in the N-terminal region, is a completely novel germline variant not detected in 6500 samples publicly available in ESP6500. In the pAML cohort (TCGA; n=197), the observations of germline variants for these telomerase complex genes were SMG6 (n=21), POT1 (n=19), NHP2 (n=1), NOP10 (n=1) GAR1 (n=1). Next, we analyzed clinical characteristics, including treatment responsiveness as assessed per modified 2006 IWG response criteria. The mean age of the 39 patients with germline telomerase machinery alterations was 67 years, 24% (9/39) were younger (age<60 years) compared to 12% (12/97) of wild type (WT; p=.12). Of note, 58% of these cases had a family history of solid tumors including breast, gastrointestinal and prostate and 8% (3/36) had a family history of myeloid malignancies. 41% (16/39) of the telomerase mutants had higher-risk MDS/sAML at presentation compared to 23% in WT cases (23/97; p=.19). A higher percentage of mutants also had complex cytogenetics compared to WT (35% vs. 13%; p=.01). Response rates to common therapies, including hypomethylating agents were similar, but we noted that none of the carrier cases (n=16) treated with lenalidomide showed therapeutic responses (0% vs. 37%). The mean overall survival of the carrier cases was lower compared to the WT (36 vs. 39 months, p=.10). When we studied cases with telomerase alterations for the presence of coinciding somatic mutations, using a targeted deep sequencing panel of the 100 most common mutations acquired in pts with germline telomerase complex alterations, we found most common the acquisition of DNMT3 (18% vs. 6%, p.10) and cohesin mutations (13% vs. 4%,p=.11). In sum, unbiased NGS sequencing approaches in MDS and related myeloid neoplasms allowed for identification of genetic germline alterations in telomerase maintenance machinery at higher rates than previously detected using targeted screening approaches, suggesting that such genetic defects may more frequently than previously thought contribute to cryptic and likely complex genetic predisposition to these diseases. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding.

Download Full-text

The Significance of the Next Generation Targeted Sequencing in the Precise Diagnosis of Pediatric Acquire Aplastic Anemia and Inherited Bone Marrow Failure Syndromes

Blood ◽

10.1182/blood.v128.22.3908.3908 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3908-3908 ◽

Cited By ~ 1

Author(s):

Wenbin An ◽

Ye Guo ◽

Yumei Chen ◽

Yao Zou ◽

Xiaojuan Chen ◽

...

Keyword(s):

Bone Marrow ◽

Family History ◽

Bone Marrow Failure ◽

Gene Mutations ◽

Chromosome Breakage ◽

Targeted Sequencing ◽

Genetic Mutations ◽

Bone Marrow Failure Syndromes ◽

Precise Diagnosis ◽

History Of

Abstract Background Diagnosis of inherited bone marrow failure syndromes (IBMFs) depend on classic clinical manifestation including early onset, physical anomalies, family history of cancer and/or bone marrow failure and chromosome breakage testing (MMC and/or DEB), mutation analyses and bone marrow chromosome analyses. At present, more than 70 pathogenic gene mutations had been identified. However, in some patients, physical anomalies is absent or delayed, and were misdiagnosed as acquired aplastic anemia(AA). Genetic analysis is very important to establish a precise diagnosis, predict cancer risk, direct treatment and genetic counseling. In this study, we focus on the application of next generation targeted sequencing in precise diagnosis of pediatric acquired AA/IBMFs, and the association between genetic abnormalities and clinical and laboratory characteristics. Methods We designed a targeted sequencing assay to test a panel of 417 genes. The panel contain reported gene associated with IBMFs and other diseases need be differentiated. Pediatic patients (≤14 year old) with suspected diagnosis of AA/IBMFs were enrolled. Peripheral blood (PB) DNA was used to genetic analysis and oral epithelia cells or PB DNA from their parents were used to identify somatic mutations and unreported polymorphism. All the results were validated by Sanger sequencing. Results The average coverage of targeted region was 98.15%, and the average sequencing depth was 315.9×. Totally, 283 patients were enrolled, including 176 clinically diagnosed acquired AA, 51 Fanconi anemia (FA), 8 dyskeratosis congenital(DKC), 30 Diamond-Blanckfan anemia(DBA), 15 congenital neutropenia(CN), and 3 congenital thrombocytopenia. Totally, 19% subjects had IBMFs related genetic mutations. In the patients who were clinically diagnosed as acquired AA patients, about 7% had IBMFs related disease-causing genetic mutations. Finally, 7 patients were genetically diagnosed as FA, 2 were DKC, 1 was WAS and 1 was SDS. In patients who were clinically diagnosed as FA, 33.4% had FANC related gene mutations. Telomere associated gene mutations were detected in 75% of clinical diagnosed DKC. For patients clinically diagnosed as DBA and CN, 36.7% and 20% were detected disease-causing mutations. After genetic screening, 2 patients who had been diagnosed as FA were modified as WAS and 1 DBA was modified as SDS. Only 26% genetic diagnosed IBMFs patients had family history of bone marrow failure, leukemia, tumor or physical anomalies. Compared with acquired AA, patients with genetic diagnosed FA were more likely to have physical anomalies of short stature and development retardation, Cafe au lait spots and finger or toe malformation(P<0.001).However, 46% patients with IBMFs did not have any type of physical anomalies. Moreover, there were only 24% patients with genetic diagnosed IBMFs had positive results of MMC induced chromosome breakage test or SCGE, and both the examinations could not differentiate subtype of IBMFs. FANCD2 mono-ubiquitination test were performed recently. However, even in the genetically confirmed FA, the positive rate was only 18% (2/11). And, there were positive results in some acquired AA patients. For FA patients with definitely genetic mutations, 62.5%(15/24) were compound heterozygous mutations,37.5%(9/24) were homozygous mutations. Mutational frequencies of FANC were: FANCA 65%, FANCD2 23%, FANCG 9%, FANCI 9% and FANCB 4%. For the mutated type, the frequencies of missense, frameshift, nonsense, splicing mutation were 42%, 26%, 16%, 16%. In our study, there were 4 undetermined patients met the clinical diagnostic criteria of FA, and having heterozygous damaged mutations in FANC genes. Conclusion In conclusion, our IBMFs associated genes targeted sequencing assay is an effective strategy for precise diagnosis of bone marrow failure diseases, especially for those without family history or physical anomalies. However, nearly half of the clinically diagnosed IBMFs patients in our study were not detected the disease-causing mutations. This may be due to the mutations in the intron area, or large fragment deletion, which cannot be detected by targeted sequencing. And the novel gene involved in IBMFs need further study. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Hepatitis-associated aplastic anemia from workout supplement: Rare but potentially fatal entity

SAGE Open Medical Case Reports ◽

10.1177/2050313x20901937 ◽

2020 ◽

Vol 8 ◽

pp. 2050313X2090193

Author(s):

Sanjog Bastola ◽

Ojbindra Kc ◽

Sumesh Khanal ◽

Alexandra Halalau

Keyword(s):

Bone Marrow ◽

Aplastic Anemia ◽

Bone Marrow Failure ◽

Liver Function Tests ◽

Marrow Transplant ◽

Clinical Syndrome ◽

Cell Transplant ◽

Hematopoietic Cell Transplant ◽

History Of ◽

Severe Pancytopenia

Hepatitis-associated aplastic anemia (HAAA) is a rare clinical syndrome characterized by bone marrow failure 1–3 months after development of hepatitis. Untreated, hepatitis-associated aplastic anemia has poor outcome and the mainstay of treatment remains either bone marrow transplant or immunosuppressive therapy. A previously healthy 21-year-old man presented with a 1-week history of right upper quadrant pain and jaundice. Admission labs revealed mixed hyperbilirubinemia and elevated transaminases ranging in 2000s IU/dl. Extensive workup for etiologies of acute hepatitis including viruses, autoimmune, toxins etc. were negative. He admitted to taking “Dust V2,” a workout supplement, for 4 months prior to the presentation. His liver function tests started to improve after conservative treatment. Two months after his discharge, he was found to have severe pancytopenia on routine labs. Bone marrow biopsy revealed hypocellular marrow consistent with aplastic anemia. Extensive workup for etiologies of aplastic anemia were negative. On literature review, none of the components of the supplement were found to cause aplastic anemia. A diagnosis of hepatitis-associated aplastic anemia was made as there was a lag time before development of anemia. His counts failed to improve despite treatment with filgrastim and he was referred for hematopoietic cell transplant.

Download Full-text

Familial Atypical Hemolytic Uremic Syndrome with Positive pS1191L (c.3572C> T) Mutation in CFH A Single-Center Experience

10.21203/rs.2.13028/v1 ◽

2019 ◽

Author(s):

Fadime ERSOY DURSUN ◽

Gözde YESIL ◽

Hasan DURSUN ◽

Gülşah SASAK

Keyword(s):

Renal Failure ◽

Chronic Renal Failure ◽

Family History ◽

Hemolytic Uremic Syndrome ◽

Atypical Hemolytic Uremic Syndrome ◽

Gene Mutations ◽

Factor H ◽

The Other ◽

History Of ◽

Uremic Syndrome

Abstract Background: Atypical hemolytic uremic syndrome is a condition characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney injury, which can exhibit a poor prognosis. Gene mutations play a key role in this disease, which may be sporadic or familial. Methods: We studied, 13 people from the same family were investigated retrospectively for gene mutations of familial atypical hemolytic uremic syndrome after a patient presented to our emergency clinic with atypical hemolytic uremic syndrome and reported a family history of chronic renal failure. Results: The pS1191L mutation in the complement factor H gene was heterozygous in 6 people from the family of the patient with atypical hemolytic uremic syndrome. One of these people was our patient with acute renal failure and the other two are followed up by the Nephrology Clinic due to chronic renal failure. The other 3 persons showed no evidence of renal failure. The index case had a history of 6 sibling deaths; two of them died of chronic renal failure. Plasmapheresis and fresh frozen plasma treatment was given to our patient. When patient showed no response to this treatment, eculizumab therapy was started. Conclusions: The study demonstrated that a thorough family history should be taken in patients with atypical hemolytic uremic syndrome. These patients may have familial type of the disease and they should be screened genetically. Eculizumab should be the first choice in the treatment with plasmapheresis. It should be kept in mind that the use of eculizumab as prophylaxis in post-transplant therapy is extremely important for prevention of rejection.

Download Full-text

DDX41 Mutations in Myeloid Neoplasms and Acute Leukemias

Blood ◽

10.1182/blood-2020-143456 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 12-13

Author(s):

Yang Zhang ◽

Fang Wang ◽

Xue Chen ◽

Hong Liu ◽

Xiaoliang Wang ◽

...

Keyword(s):

Family History ◽

Germline Mutation ◽

Mrna Splicing ◽

The Other ◽

Biallelic Mutation ◽

Acute Leukemias ◽

Myeloid Neoplasms ◽

Uncertain Significance ◽

History Of ◽

The Dead

DDX41 is thought to be a tumor suppressor gene involved in pre-mRNA splicing, innate immunity and rRNA processing. Myeloid neoplasms with germline DDX41 mutations have been included as a new diagnostic category in the 2016 WHO classification. However, there are limited studies describing the mutation profile of myeloid neoplasms and acute leukemias associated with DDX41 mutation. We analyzed the prevalence and characteristics of DDX41 mutations in an unselected cohort of 1764 patients with myeloid neoplasms and acute leukemias, including 720 subjects with AML, 91 with MDS, 41 with MPN, 16 with MDS/MPN, 760 with ALL, and 42 with MPAL. Next-generation sequencing was performed on 86 genes closely related to hematologic neoplasms. The fingernail specimens or blood samples in remission were taken as control samples to verify the mutation from possible germline sources. We identified 21 different DDX41 mutations in 16 unrelated patients (6 MDS/AML, 1 CMML, 9 ALL) that were classified as causal (n=17) and uncertain significance (n=4) variants. The acquisition of a somatic DDX41 mutation was also considered as a very strong criterion for causality, the uncertain significance variants were excluded. Nine causal variants have not been reported. 53% of variants were located on the DEAD domain and 24% on the Helicase C domain, the rest were located upstream of the DEAD domain. Ten variants were germline that the majority (80%) were located upstream of the Helicase C domain, 7 variants were somatic and were scattered. In 6 patients with MDS/AML and DDX41 mutations, the median age was 49 years (range, 28-78y) and 57% were male. None of the patients had del 5/5q. Five (83%) patients had personal history of cytopenia prior to MDS/AML diagnosis, while only one patient had a family history of anemia and one patient's aunt died of leukemia. Four (67%) patients harbor DDX41 germline/somatic biallelic mutation, two with typical biallelic mutation (N-terminal germline nonsense and C-terminal somatic missense), the other two with atypical biallelic mutation (N-terminal germline missense and C-terminal somatic missense). The average age of patients with DDX41 atypical biallelic mutation (48y) seems lower than that with typical biallelic mutation (74y). The rest two patients harbor single germline mutations and one of them concomitant with SF3B1 mutation, which is a component of spliceosome complex also involving in mRNA splicing. DDX41 mutations were identified in 7 patients with B-ALL and one with T-ALL. The median age was 9 years (range, 4-2 y) and 56% were male. None of the patients had a family history of hematological malignancy and del 5/5q. Unlike in myeloid neoplasms, no DDX41 biallelic mutations were identified that 5 patients had single somatic mutation (3 missenses, 1 nonsense) and 4 had single germline mutation (all are missenses). Among MDS/AML patients with DDX41 biallelic mutation, only one received treatment who relapsed after HSCT and received second HSCT, the time of overall survival (OS) was 74 months, the other 3 quite after diagnosed. In patients with MDS/AML and DDX41 single germline mutation, one received 4 courses of treatment with decitabine and half-dose CAG regimen, then transformed to AML and abandoned, the other one received 10 courses of chemotherapy and showed continuous no remission. The time of OS was 17 and 31 months, respectively. Among ALL patients with DDX41 single somatic mutation, 80% (4/5) received HSCT, 80% (4/5) were in complete remission (CR), one died of post-transplant infection, the median OS was 25 months. Among ALL patients with DDX41 single germline mutation, all the three patients received HSCT and were in CR, the median OS was 37 months. The genotype-phenotype correlations regarding germline DDX41 mutations should be clarified more specifically, the most prevalent loss of function mutations, predisposes to myeloid disease at the same age as sporadic disease, whereas point mutations in the DEAD domain (this study) or helicase C domain (previous report) were speculated to cause earlier onset disease. Moreover, this study reported for the first time that DDX41 mutations have also been found in ALL, which expanded its phenotypic spectrum. The characteristics of DDX41 mutation in ALL are different from myeloid neoplasm, the age of onset is young, and no germline/somatic biallelic mutation have been observed, suggesting that it might be involved in different pathogenesis mechanisms. Disclosures No relevant conflicts of interest to declare.

Download Full-text

The High Frequency of HLA-A*0206 Allele in Japanese Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) and Its Significance.

Blood ◽

10.1182/blood.v104.11.3703.3703 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3703-3703

Author(s):

Kazuhiko Ikeda ◽

Tsutomu Shichishima ◽

Kazuko Akutsu ◽

Yukio Maruyama

Keyword(s):

Bone Marrow ◽

High Frequency ◽

Past History ◽

Bone Marrow Failure ◽

Japanese Patients ◽

Negative Group ◽

Positive Group ◽

Hla Dr ◽

Transfusion Requirements ◽

History Of

Abstract PNH is an acquired hematologic disorder which is characterized by complement-mediated hemolysis, thrombosis, and bone marrow failure. Also, PNH is one disorder of bone marrow failure syndromes, including aplastic anemia (AA) and myelodysplastic syndrome (MDS). It is well known that immunologic mechanisms by cytotoxic T lymphocytes (CTLs) contribute to pathophysiology of these disorders. In fact, some reports (Maciejewski et al, Blood, 2001; Shichishima et al, Blood, 2002) showed that HLA-DR*1501 is related with clinical pathophysiology of PNH. In this study, to clarify significance of CD8+ CTLs in pathophysiology of PNH, we investigated HLA class I (A and B) alleles in Japanese patients with PNH (female: male=7:17), AA (female: male=14:15), and MDS (female: male=6:16) by high-resolution method using polymerase-chain reaction after obtaining informed consent and approval from the institutional Human Research Committee. Mean age ± standard deviation of PNH, AA, and MDS patients was 52 ± 16, 54 ± 20, and 59 ± 18, respectively. HLA genotyping showed that the frequency of HLA-A*0206 allele in PNH patients (22.9%) was significantly different from those in 309 unrelated Japanese individuals (Saito et al, Tissue Antigens, 2000) (7.7%; p<0.02) and AA patients (5.2%; p<0.01). In contrast, we found no significant differences in the frequencies of the other alleles between PNH, AA, or MDS patients and the controls or between these disorders, except for high frequency of HLA-B40 alleles in AA patients (Nakamura et al, Blood, 2003). Then, various clinical parameters, including peripheral blood, bone marrow blood, and laboratory findings, proportions of glycosylphosphatidylinositol protein-negative population in erythrocytes, granulocytes, and monocytes, findings of chromosomal analyses and HLA-DR alleles, transfusion requirements, past history of AA, and history of thrombosis, were statistically compared between HLA-A*0206-positive group (n=10) and -negative group (n=14) in PNH patients. Statistical analyses showed that the reticulocyte counts , the values of lactate dehydrogenase, and the frequency of PNH patients with over 30% of CD59− erythrocytes in HLA-A*0206-positive group were significantly higher than in HLA-A*0206-negative group (121 ± 49 x 109/L vs 76.9 ± 43.9 x 109/L, p<0.03; 2866 ± 2606 IU/L vs 938 ± 775, p<0.02; and 80.0 % vs 28.6 %, p<0.02, respectively). Moreover, we found no AA (n=3) and MDS (n=5) patients with both the HLA-A*0206 allele and more than 1% of CD59− granulocytes. In conclusion, our findings suggest that the HLA-A*0206 allele in PNH may be correlated with the grade of the disease by complement-mediated hemolysis during negative selection of PNH clones, probably due to immunologic mechanisms by CD8+ CTLs.

Download Full-text

Intermittent Low Dose Interleukin 11 Can Lead to Transfusion Independence in Patients with Chronic Myelo-Monocytic Leukemia and Thrombocytopenia.

Blood ◽

10.1182/blood.v104.11.3939.3939 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3939-3939

Author(s):

Chirag Shah ◽

Teresa C. Gentile

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Platelet Transfusion ◽

Low Dose ◽

Transfusion Requirement ◽

Bone Marrow Failure ◽

Monocytic Leukemia ◽

Significant Toxicity ◽

History Of ◽

Interleukin 11

Recombinant Interleukin- 11 (IL-11) is a thrombopoietic cytokine that stimulates megakaryocytopoiesis in vitro and platelet production in vivo. It attenuates post chemotherapy thrombocytopenia at a dose of 50 mcg/ kg/ day subcutaneously (SC). Unfortunately, prolonged administration is associated with significant toxicity including peripheral and pulmonary edema at this dose. Administration of low dose IL-11 at 10 mcg/kg/day SC has shown efficacy in bone marrow failure states without significant toxicity. We report two cases of chronic myelo-monocytic leukemia (CMML) with transfusion dependent thrombocytopenia who received intermittent low dose IL-11 without significant toxicity. Case Reports- Patient 1 is a 79-year-old male with history of CMML with pancytopenia of three years duration. Recently he required transfusion of platelets with his platelet counts falling to less than 15 x 109/L. His cytogenetic study showed normal karyotype, 46 XY. He required platelet transfusion at every 14 days. He initially was started on IL-11 at 10mcg/kg/day, 5 days per week.. His platelet count increased to above 30 x 109/L and he became transfusion independent within two weeks. Unfortunately on this schedule he developed edema and mild CHF. IL-11 was stopped for two weeks and upon resolution of toxicity, restarted at 10 mcg/kg/day on Monday, Wednesday and Friday. He has remained transfusion independent without recurrence of edema at 5 months on this schedule. Patient 2 was a 63-year-old male with previous history of chronic lymphocytic leukemia and diffuse large B cell lymphoma who developed CMML with severe pancytopenia. His karyotype was 46, XY, −7, +21. His platelet count was consistently less than 10 x 109/L. He required platelet transfusion twice a week. He was started on IL-11 at 10mcg/kg/day for 5 days per week, two weeks on and two weeks off. His platelet count increased to as high as 64 x 109/L after 2nd cycle. His platelet transfusion requirement decreased from every 3rd day to every 10th-14th day. He experienced no peripheral or pulmonary edema. Conclusion: Administration of low dose IL-11 in other bone marrow failure states has been reported but its use has not been described in CMML. Our observation in these 2 patients suggests that IL-11 has efficacy in CMML and is very well tolerated at low doses on an intermittent administration schedule. IL-11 may decrease the transfusion requirement in transfusion dependent patients. Further studies are needed to evaluate overall impact on larger number of patients who require regular platelet transfusion.

Download Full-text

The Role of Telomerase Activity and Telomere Length in Cellular Sensitivity to DNA Damaging Agents.

Blood ◽

10.1182/blood.v108.11.4192.4192 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4192-4192

Author(s):

Greg T. Rice ◽

Michael A. Beasley ◽

Ike I. Akabogu ◽

Erik R. Westin ◽

Dale A. Winnike ◽

...

Keyword(s):

Bone Marrow ◽

Telomere Length ◽

Bone Marrow Failure ◽

Skin Pigmentation ◽

Cytotoxic Agents ◽

Cell Surface Expression ◽

Cell Transplant ◽

Telomere Shortening ◽

Hematopoietic Stem ◽

Increased Sensitivity

Abstract Dyskeratosis congenita (DC) is a premature aging syndrome characterized by progressive bone marrow failure, abnormal skin pigmentation and nail dystrophy. We have described an autosomal dominant form of DC (AD DC) in a large three-generation kindred that is due to a mutation in the gene encoding human telomerase RNA (hTR). While telomere shortening is a normal consequence of the aging process, DC patients display extremely short telomeres in many somatic cell types, including hematopoietic cells, and they often suffer from bone marrow failure. Allogeneic hematopoietic stem cell transplant (HSCT) remains the only curative therapy for marrow failure in DC. However, HSCT in DC is generally poorly tolerated and associated with significant morbidity, perhaps as a consequence of increased sensitivity of dividing cells to cytotoxic agents during myeloablative therapy. To test this hypothesis, we characterized lymphocytes from various AD DC patients and age matched controls that had been placed in long term culture following in vitro exposure to irradiation (137Cs) and varying doses of Taxol, Adriamycin, and Etoposide. Cell proliferation and viability were quantified by direct visual counting on a hemocytometer, and flow cytometry was employed to assess apoptosis and cell surface expression of senescent markers. In addition to DC lymphocytes having a decreased proliferative capacity and higher basal apoptotic levels, an increased sensitivity to irradiation, Taxol, Adriamycin, and Etoposide was noted. These results suggest that telomere shortening may be an important factor in determining cellular tolerance to cytotoxic therapy and support the concept of reduced intensity HSCT regimens in both aged individuals and DC patients. Further studies have been initiated to determine whether reconstitution of telomere length in DC cells alters response to cytotoxic agents.

Download Full-text

Age-Adjusted Telomere Length Is Not Associated with Variation in Hematological Parameters in Ageing Women

Blood ◽

10.1182/blood.v112.11.3102.3102 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3102-3102

Author(s):

Isabelle Fleury ◽

Sylvie Provost ◽

Claude Belisle ◽

Lambert Busque

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

Telomere Length ◽

Marrow Transplantation ◽

Copy Number ◽

Bone Marrow Failure ◽

Telomere Maintenance ◽

Hematological Parameter ◽

Allogeneic Bone ◽

Potential Biomarker

Abstract Background. Telomeres play a crucial role in maintaining physical integrity of chromosomes. In the absence of telomerase, telomere length (TL) shortens with each cell division up to a critical threshold where cellular senescence occurs. TL is inversely correlated with age, is longer in women than in men, and demonstrates a strong heritability. Normal blood counts are maintained through out life by an extraordinary number of cell divisions rendering telomere maintenance primordial to prevent stem cell exhaustion. In fact, some cases of bone marrow failure syndromes, such as aplastic anemia and dyskeratosis congenital, have been linked to mutation in the telomerase gene; and stressed hematopoiesis, such at it occurs during the first year following allogeneic bone marrow transplantation induces TL shortening. We hypothesized that individuals with shorter TL may have lower blood counts and a decreased bone marrow reserve. The evaluation of TL as a potential biomarker of ageing hematopoiesis is important in the context of bone marrow transplantation performed with increasingly old donors. Methods. We measured TL in 1583 women, predominantly aged over 60, all originating from 288 French-Canadian families using a real-time quantitative PCR method that measures the number of telomere repeats relatively to the copy number of a single copy number gene. Telomeres were adjusted for age. Pearson or Spearman correlations were used to determine association between age-adjusted TL (aTL) and hematological parameter according to, respectively, whether or not a normal distribution was observed for these data. A Bonferroni correction was further applied to set the statistical significance threshold. Results. aTL varied significantly between individuals of the cohort, but no correlation was detected with hemoglobin levels (−0,001; p=0,978), mean corpuscular volume (−0,031; p=0,403); leucocytes (0,055; p=0,139); neutrophils (0,078; p=0,036), monocytes, (0,059; p=0,113), eosinophils (−0,032; p=0,394) and platelets (0,030; p=0,428) counts. Conclusion. Based on our analysis, TL do not predict blood cells counts in ageing women and may not be a useful biomarker for donor selection. This could also suggest that there is a threshold beyond which TL has an effect on hematopoiesis and that point was not reached in our cohort.

Download Full-text

Gene Expression Profiling Distinguishes Waldenstrom's Macroglobulinemia Patients Presenting with Familial Disease, Advanced IPSS Prognostic Score, and Previous Treatment with Rituximab.

Blood ◽

10.1182/blood.v114.22.3930.3930 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3930-3930 ◽

Cited By ~ 1

Author(s):

Zachary Hunter ◽

Evdoxia Hatjiharissi ◽

Jenny Sun ◽

Yang Cao ◽

Hsiuyi Tseng ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Family History ◽

B Cell ◽

G Protein ◽

Expression Profiling ◽

Prognostic Score ◽

G Protein Coupled Receptors ◽

History Of ◽

G Protein Coupled

Abstract Abstract 3930 Poster Board III-866 Background The use of gene expression profiling (GEP) was used to dissect the molecular profile of Waldenstrom's macroglobulinemia. Bone marrow CD19+ cells from 22 WM patients and 8 healthy donor (HD) were used in these studies, with application of analytics geared toward non-normally distributed data. Patient characteristics were as follows: median age 64 years; bone marrow disease involvement 35%; serum IgM 3,295 mg/dl; beta-2 microglobulin (B2M) 2.7 mg/L; WM ISS Prognostic Score 2. Four patients (18%) previously received rituximab, and 4 (18%) patients had a family history of WM and/or related B-cell disorders. Materials and Methods GEP was performed using the Affymetrix U133 plus 2 platform on CD19+ selected, CD138 depleted bone marrow cells. Array quality checks, normalization, and unsupervised hierarchical clustering were conducted using dChip (Li and Wong 2001 PNAS). These results were then used for further analysis via custom perl scripts that used 10,000 resampled groups to calculate bootstrap percentile based 95% confidence intervals (CI) for both mean and median values. Comparisons between groups were evaluated using approximate permutation testing. To help identify potential biomarkers, absence/presence calls from DCHIP based on the perfect match vs. mismatch comparisons were tabulated for each group and the contingency table resulting from group comparisons were analyzed using a Fisher's exact test. A gene was considered significant if 50% of its probes displayed at least a 2-fold change, mutual exclusion of means/median values and respective 95% CI, and p < 0.01 for both mean and median comparisons. This data was then compared with dChip clustering results and analyzed using Ingenuity Pathway Analysis (Ingenuity Systems). Results Significantly down regulated genes included DLL1 (-13.5 fold, expressed 0% WM vs. 88% HD, P<0.0001), LILRB5 (-13.9 fold expressed in 5% WM vs. 62% HD, P=0.003), MXD1 (-10.3 fold), FOSL2 (-8.8 fold), CXCL12 (-8.0 fold), and ATF3 (-7.5 fold). Up-regulated genes included a number of G-protein coupled receptors including LPAR5 (+7.3 fold), CYSLTR1 (+6.8 fold), and GPER (+16 fold). Other genes of interest included TLR9 (+3.9 fold), TLR10 (+2.8 fold), along with several anti-viral proteins including RANSEL (+6.9 fold), OAS1 (+7.8 fold), and OAS2 (+2.3 fold). Subgroup analysis revealed an up regulation of GP5 (+3.5 fold), LHX1 (+3.3 fold), ERG1 (+3.2 fold), FZD1 (+2.6 fold), and EFNB2 (+2.2 fold) in patients with a family history of WM and/or related B-cell disorders. For those with a high ISS score (≥3), we observed differences in WNT5A (+5.04 fold), CXCL12 (+3.5 fold), NOTCH4 (-2.6 fold) and IL2RA (-2.6 fold). Lastly, WM patients previously treated with rituximab displayed increased expression of BTG2 (+2.3 fold), MCL2 (+2.5 fold), and ARMCX2 (+5.5 fold). Conclusions The results of these studies demonstrate differential expression of several novel genes in WM including g protein coupled receptors and genes involved in interferon signaling. Importantly, these studies demonstrate for the first time differential expression of several gene candidates involved in B-cell differentiation that distinguish sporadic versus familial WM. Moreover, GEP revealed a unique profile for patients presenting with poor prognostic disease. Lastly, these studies reveal the up-regulation of 2 tumor suppressor genes, and the anti-apoptotic gene MCL-2 in WM patients treated with rituximab. The findings of these studies therefore have important implications in the pathogenesis, prognostication and treatment of WM. Disclosures: No relevant conflicts of interest to declare.

Download Full-text