Rodrigo T. Calado

Direct Comparison of Flow-FISH and qPCR as Diagnostic Tests for Telomere Length Measurement in Humans

Telomere length measurement is an essential test for the diagnosis of telomeropathies, which are caused by excessive telomere erosion. Commonly used methods are terminal restriction fragment (TRF) analysis by Southern blot, fluorescence in situ hybridization coupled with flow cytometry (flow-FISH), and quantitative PCR (qPCR). Although these methods have been used in the clinic, they have not been comprehensively compared. Here, we directly compared the performance of flow-FISH and qPCR to measure leukocytes telomere length of healthy individuals and patients evaluated for telomeropathies, using TRF as standard. TRF and flow-FISH showed good agreement and correlation in the analysis of healthy subjects (R2u200a=u200a0.60; p<0.0001) and patients (R2u200a=u200a0.51; p<0.0001). In contrast, the comparison between TRF and qPCR yielded modest correlation for the analysis of samples of healthy individuals (R2u200a=u200a0.35; p<0.0001) and low correlation for patients (R2u200a=u200a0.20; pu200a=u200a0.001); Bland-Altman analysis showed poor agreement between the two methods for both patients and controls. Quantitative PCR and flow-FISH modestly correlated in the analysis of healthy individuals (R2u200a=u200a0.33; p<0.0001) and did not correlate in the comparison of patients samples (R2u200a=u200a0.1, pu200a=u200a0.08). Intra-assay coefficient of variation (CV) was similar for flow-FISH (10.8±7.1%) and qPCR (9.5±7.4%; pu200a=u200a0.35), but the inter-assay CV was lower for flow-FISH (9.6±7.6% vs. 16±19.5%; pu200a=u200a0.02). Bland-Altman analysis indicated that flow-FISH was more precise and reproducible than qPCR. Flow-FISH and qPCR were sensitive (both 100%) and specific (93% and 89%, respectively) to distinguish very short telomeres. However, qPCR sensitivity (40%) and specificity (63%) to detect telomeres below the tenth percentile were lower compared to flow-FISH (80% sensitivity and 85% specificity). In the clinical setting, flow-FISH was more accurate, reproducible, sensitive, and specific in the measurement of human leukocytes telomere length in comparison to qPCR. In conclusion, flow-FISH appears to be a more appropriate method for diagnostic purposes.

Telomeres and marrow failure

Telomeres, repeat sequences at the ends of chromosomes, are protective chromosomal structures highly conserved from primitive organisms to humans. Telomeres inevitably shorten with every cell cycle, and telomere attrition has been hypothesized to be fundamental to normal senescence of cells, tissues, and organisms. Molecular mechanisms have evolved to maintain their length and protective function; telomerase (TERT) is a reverse transcriptase enzyme that uses an RNA molecule (TERC) as the template to elongate the 3 ends of telomeres. Shelterin is a collection of DNA-binding proteins that cover and protect telomeres. The recent discovery of inherited mutations in genes that function to repair telomeres as etiologic in a range of human diseases, which have clinical manifestations in diverse tissues, including the hematopoietic tissue, suggests that defects in telomere repair and protection can cause organ failure. Dyskeratosis congenita is the prototype of telomere diseases; it is characterized by bone marrow failure, mucocutaneous abnormalities, pulmonary fibrosis, liver cirrhosis, and increased susceptibility to cancer, including acute myeloid leukemia. Aplastic anemia, acute myeloid leukemia, and idiopathic pulmonary fibrosis also are associated with inherited mutations in telomere repair or protection genes. Additionally, telomere defects associate with predisposition to hematologic malignancy and epithelial tumors. Telomere erosion is abnormally rapid in patients with mutations in telomerase genes but also after hematopoietic stem cell transplant, and telomeres are naturally shorter in older individuals-all conditions associated with higher rates of malignant diseases. In human tissue culture, short telomeres produce end-to-end chromosome fusion, nonreciprocal translocations, and aneuploidy.

HFE gene mutations in coronary atherothrombotic disease

Although iron can catalyze the production of free radicals involved in LDL lipid peroxidation, the contribution of iron overload to atherosclerosis remains controversial. The description of two mutations in the HFE gene (Cys282Tyr and His63Asp) related to hereditary hemochromatosis provides an opportunity to address the question of the association between iron overload and atherosclerosis. We investigated the prevalence of HFE mutations in 160 survivors of myocardial infarction with angiographically demonstrated severe coronary atherosclerotic disease, and in 160 age-, gender- and race-matched healthy control subjects. PCR amplification of genomic DNA followed by RsaI and BclI restriction enzyme digestion was used to determine the genotypes. The frequency of the mutant Cys282Tyr allele was identical among patients and controls (0.022; carrier frequency, 4.4%), whereas the mutant His63Asp allele had a frequency of 0.143 (carrier frequency, 27.5%) in controls and of 0.134 (carrier frequency, 24.5%) in patients. Compound heterozygotes were found in 2 of 160 (1.2%) controls and in 1 of 160 (0.6%) patients. The finding of a similar prevalence of Cys282Tyr and His63Asp mutations in the HFE gene among controls and patients with coronary atherothrombotic disease, indirectly questions the possibility of an association between hereditary hemochromatosis and atherosclerosis.

Telomere attrition and candidate gene mutations preceding monosomy 7 in aplastic anemia

The pathophysiology of severe aplastic anemia (SAA) is immune-mediated destruction of hematopoietic stem and progenitor cells (HSPCs). Most patients respond to immunosuppressive therapies, but a minority transform to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), frequently associated with monosomy 7 (-7). Thirteen SAA patients were analyzed for acquired mutations in myeloid cells at the time of evolution to -7, and all had a dominant HSPC clone bearing specific acquired mutations. However, mutations in genes associated with MDS/AML were present in only 4 cases. Patients who evolved to MDS and AML showed marked progressive telomere attrition before the emergence of -7. Single telomere length analysis confirmed accumulation of short telomere fragments of individual chromosomes. Our results indicate that accelerated telomere attrition in the setting of a decreased HSPC pool is characteristic of early myeloid oncogenesis, specifically chromosome 7 loss, in MDS/AML after SAA, and provides a possible mechanism for development of aneuploidy.

Exome sequencing reveals a thrombopoietin ligand mutation in a Micronesian family with autosomal recessive aplastic anemia.

We recently identified 2 siblings afflicted with idiopathic, autosomal recessive aplastic anemia. Whole-exome sequencing identified a novel homozygous missense mutation in thrombopoietin (THPO, c.112C>T) in both affected siblings. This mutation encodes an arginine to cysteine substitution at residue 38 or residue 17 excluding the 21-amino acid signal peptide of THPO receptor binding domain (RBD). THPO has 4 conserved cysteines in its RBD that form 2 disulfide bonds. Our in silico modeling predicts that introduction of a fifth cysteine may disrupt normal disulfide bonding to cause poor receptor binding. In functional assays, the mutant-THPO-containing media shows two- to threefold reduced ability to sustain UT7-TPO cells, which require THPO for proliferation. Both parents and a sibling with heterozygous R17C change have reduced platelet counts, whereas a sibling with wild-type sequence has normal platelet count. Thus, the R17C partial loss-of-function allele results in aplastic anemia in the homozygous state and mild thrombocytopenia in the heterozygous state in our family. Together with the recent identification of THPO receptor (MPL) mutations and the effects of THPO agonists in aplastic anemia, our results have clinical implications in the diagnosis and treatment of patients with aplastic anemia and highlight a role for the THPO-MPL pathway in hematopoiesis in vivo.

Age-related changes of the multidrug resistance P-glycoprotein function in normal human peripheral blood T lymphocytes

The multidrug resistance P-glycoprotein is a transmembrane efflux pump expressed by lymphocytes and is involved in their cytolytic activity. In the present study, we investigated the age-related changes of P-glycoprotein function in normal peripheral blood lymphocytes. Blood samples from 90 normal volunteers (age range, 0 to 86 years) were analyzed. P-glycoprotein function was assessed by the flow cytometric rhodamine 123 assay. P-glycoprotein function was highest in cord blood and progressively declined with age in peripheral blood T CD4+ and CD8+ cells. In contrast, P-glycoprotein function did not vary with age in CD19+ B or CD16+CD56+ natural killer cells. These data suggest that the decline in P-glycoprotein function in T CD4+ and CD8+ lymphocytes as a function of age may contribute to the decrease in T cell cytolytic activity with aging.

Age‐related changes of immunophenotypically immature lymphocytes in normal human peripheral blood

The presence of phenotypically immature lymphocytes in umbilical cord blood has been a controversial topic. Moreover, their changes with age have not been systematically evaluated. In the present study, relative and absolute numbers of CD34+, CD10+CD19+, and CD4+CD8+ cell subsets were determined in umbilical cord blood from 12 full-term normal newborns, 43 children aged 1 month to 6 years, and 10 young adults. The samples were processed by whole-blood lysis and monoclonal antibody staining, and cells were analyzed by flow cytometry. Immature cells were present in cord blood and progressively declined in both absolute and percentage numbers with age, each according to a particular curve, reaching youth values roughly at age 2-4 years. These results demonstrate that phenotypically immature cells normally circulate at low levels in peripheral blood, mostly at birth and during infancy, but also during youth.

Higher Expression of Transcription Targets and Components of the Nuclear Factor‐κB Pathway Is a Distinctive Feature of Umbilical Cord Blood CD34+ Precursors

Delayed engraftment, better reconstitution of progenitors, higher thymic function, and a lower incidence of the graft‐versus‐host disease are characteristics associated with umbilical cord blood (UCB) transplants, compared with bone marrow (BM). To understand the molecular mechanisms causing these intrinsic differences, we analyzed the differentially expressed genes between BM and UCB hematopoietic stem and progenitor cells (HSPCs). The expressions of approximately 10,000 genes were compared by serial analysis of gene expression of magnetically sorted CD34+ cells from BM and UCB. Differential expression of selected genes was evaluated by real‐time polymerase chain reaction on additional CD34+ samples from BM (n = 22), UCB (n = 9), and granulocyte colony stimulating factor‐mobilized peripheral blood (n = 6). The overrepresentation of nuclear factor‐κB (NF‐κB) pathway components and targets was found to be a major characteristic of UCB HSPCs. Additional promoter analysis of 41 UCB‐overrepresented genes revealed a significantly higher number of NF‐κB cis‐regulatory elements (present in 22 genes) than would be expected by chance. Our results point to an important role of the NF‐κB pathway on the molecular and functional differences observed between BM and UCB HSPCs. Our study forms the basis for future studies and potentially for new strategies to stem cell graft manipulation, by specific NF‐κB pathway modulation on stem cells, prior to transplant.

Aplastic anaemia and telomerase RNA mutations

management of patients with minor illnesses in general practice: multicentre, randomised controlled trials. BMJ 2000; 320: 1038–43. 3 Venning P, Durie A, Roland M, Roberts C, Leese B. Randomised controlled trial comparing cost effectiveness of general practitioners and nurse practitioners in primary care. BMJ 2000; 320: 1048–53. 4 Horrocks S, Anderson E, Salisbury C. Systematic review of whether nurse practitioners working in primary care can provide equivalent care to doctors. BMJ 2002; 324: 819–23. Aplastic anaemia and telomerase RNA mutations

Intravenous infusion of allogeneic mesenchymal stromal cells in refractory or relapsed aplastic anemia

BACKGROUND AIMSnFor patients with aplastic anemia (AA) who are refractory to anti-thymocyte globulin (ATG) and cyclosporine, a second course of immunosuppression is successful in only one-fourth to one-third of cases.nnnMETHODSnWe conducted a phase 1/2 study to evaluate the addition of two to five weekly intravenous infusions of allogeneic unrelated non-human leukocyte antigen-matched bone marrow-derived mesenchymal stromal cells (MSCs) (median, 2.7xa0× 10(6) cells/kg/infusion; range, 1.3-4.5) to standard rabbit ATG and cyclosporine in nine patients with refractory or relapsed AA.nnnRESULTSnAfter a median follow-up of 20 months, no infusion-related adverse event was observed, but four deaths occurred as the result of heart failure and bacterial or invasive fungal infections; only two patients achieved partial hematologic responses at 6 months. We failed to demonstrate by fluorescence in situ hybridization or variable number tandem repeat any MSC engraftment in patient marrow 30, 90 or 180 days after infusions.nnnCONCLUSIONSnInfusion of allogeneic MSCs in AA is safe but does not improve clinical hematologic response or engraft in recipient bone marrow. This study was registered at clinicaltrials.gov, identifier: NCT01297972.