Seroprevalence and Trends of HTLV-1/2 among Blood Donors of Santo Domingo, Dominican Republic, 2012-2017.

OBJECTIVE: Being a Caribbean country, the Dominican Republic is considered endemic for HTLV-1. Viral screening in blood banks is recommended for this blood borne infection. The purpose of this work is to analyze the seroprevalence and trends of HTLV-1/2 in the Dominican Republic blood donors; it is focused on Santo Domingo, the capital of the country, which has the largest blood donation activity. We also aim at comparing our findings with published data from neighboring countries. METHODS: We performed a retrospective cross-sectional study of 10 blood centers of Santo Domingo, which reported HTLV and the other blood-transmitted infections in full. They represent more than 40% of the province's blood donations. Annual seroprevalence of HTLV-1/2, period prevalence (2012-2017), and time trend were determined. RESULTS: A total of 352,960 blood donations were evaluated. The HTLV-1/2 period prevalence was 0.26% (929/352,960)(95% CI: 0.24-0.28%). We also found a marked predominance of replacement donation (90.4%) in comparison to voluntary contributions (9.6%). Therefore, this blood donor study may provide clues on the general prevalence of the infection. CONCLUSIONS: Seroprevalence of HTLV-1/2 in blood donors of Santo Domingo, Dominican Republic, showed a relatively low and steady trend in the studied period.

Haemoglobinopathies with high oxygen affinity. Experience of Erythropathology Cooperative Spanish Group.

Haemoglobinopathies are the world's most frequently found monogenic disorders. In the cases with high oxygen affinity, the decrease in the liberation of the oxygen determines a secondary erythrocytosis. In this work, we present 17 unrelated families of Caucasian race and of Spanish origin, with ten variants of haemoglobin or haemoglobinopathies with high oxygen affinity which were diagnosed in our laboratory. Of the ten haemoglobinopathies, in four (the Hb San Diego, the Hb Johnstown, the Hb Malmö and the Hb Columbia-Missouri), the change of amino acid affects zones of the contact alpha(1)beta(2); in two variants (the Hb Strasbourg and the Hb Syracuse), it affects the unions with 2,3-DPG in the central cavity; in the other two (the Hb Badalona and the Hb La Coruña), the cavity of contact with the group haem is affected; in one (Hb Bethesda), it affects the zone of contact alpha(1)beta(1;) and in one (Hb Olympia), the position 20 of the chain in the helix B in the surface of the protein is affected. In all cases, the change of amino acid, though of different form, facilitates that the quaternary structure of the haemoglobin becomes stable in its relaxed configuration so the transfer of oxygen and the P(50) value are decreased. All cases were sent to our laboratory because of shown erythrocytosis. In the majority of them, the diagnosis was done during an analysis of routine or for being relatives of the first ones.

HTLV-1 infection: An emerging risk. Pathogenesis, epidemiology, diagnosis and associated diseases.

The Human T-Lymphotropic Virus type 1 (HTLV-1) affects up to 10 million people worldwide. It is directly associated to one of the most aggressive T cell malignancies: Adult T Cell Leukemia-Lymphoma (ATLL) and a progressive neurological disorder, Tropical Spastic Paraparesis/ HTLV-1 Associated Myelopathy (TSP/HAM). Also, infected patients tend to have more severe forms of infectious diseases such as Strongyloidiasis and Tuberculosis. HTLV spreads through parenteral, sexual, and vertical (mother-to-child) routes. Effective viral transmission is produced mainly by cell to cell mechanism, unlike other retroviruses such as HIV, which usually spread infecting cells in a cell-free form. HTLV also has a peculiar distribution, with clusters of high endemicity in nearby areas of very low prevalence or absence of the virus. This could be explained by factors including a possible founder effect, the predominance of mother to child transmission and the cell-to-cell trans-mission mechanisms. More data on viral epidemiology are needed in order to develop strategies in endemic areas aimed at reducing viral dissemination. In this review, we critically analyze HTLV-1 pathogenesis, epidemiology, diagnosis, associated diseases, preventive strategies, and treatments, with emphasis to the emerging risk for Europe and particularly Spain, focusing on prevention methods to avoid viral transmission and associated diseases.

Understanding alpha-globin gene regulation: Aiming to improve the management of thalassemia.

Over the past 50 years, many advances in our understanding of the general principles controlling gene expression during hematopoiesis have come from studying the synthesis of hemoglobin. Discovering how the alpha- and beta-globin genes are normally regulated and documenting the effects of inherited mutations that cause thalassemia have played a major role in establishing our current understanding of how genes are switched on or off in hematopoietic cells. Previously, nearly all mutations causing thalassemia have been found in or around the globin loci, but rare inherited and acquired trans-acting mutations are being found more often. Such mutations have demonstrated new mechanisms underlying human genetic disease. Furthermore, they are revealing new pathways in the regulation of globin gene expression that, in turn, may open up new avenues for improving the management of patients with common types of thalassemia.

Preliminary experience in external quality control of RT-PCR PML-RAR alpha detection in promyelocytic leukemia.

To standardize the results obtained in PML/RAR alpha RT-PCR detection by laboratories of hospitals involved in the Spanish Program for Treatment of Hematological Malignancies (PETHEMA) LPA-96, designed for the treatment of acute promyelocytic leukemia (APL), cDNA samples obtained by reverse transcription of RNA from bone marrow samples of patients with APL were sent to participating laboratories. During the first year of this external quality assessment trial nine samples were tested by a maximum of 12 laboratories. The control gene was satisfactorily amplified in 90% of the samples (62 of 69 samples), supporting the adequacy of the cDNA to be used as control sample. There was an 83% concordance between laboratories for PML/RAR alpha detection with similar results for the type of PML/RR alpha rearrangements. However, 17% disagreement still remained, attributable to low sensitivity or inadequacy of methods followed. The results stressed the need for implementation of an external quality assessment scheme to ensure the standardization of the results.

Monosomy for the most telomeric, gene-rich region of the short arm of human chromosome 16 causes minimal phenotypic effects.

We have examined the phenotypic effects of 21 independent deletions from the fully sequenced and annotated 356 kb telomeric region of the short arm of chromosome 16 (16p13.3). Fifteen genes contained within this region have been highly conserved throughout evolution and encode proteins involved in important housekeeping functions, synthesis of haemoglobin, signalling pathways and critical developmental pathways. Although a priori many of these genes would be considered candidates for critical haploinsufficient genes, none of the deletions within the 356 kb interval cause any discernible phenotype other than alpha thalassaemia whether inherited via the maternal or paternal line. These findings contrast with previous observations on patients with larger (> 1 Mb) deletions from the 16p telomere and therefore address the mechanisms by which monosomy gives rise to human genetic disease.

Deletion of BCR region 3' in chronic myelogenous leukemia.

The t(9;22)(q34;q11) produces the BCR/ABL fusion gene which codifies a 210 kb protein with a strong tyrosine kinase activity and is involved in cellular development and growth. Because this translocation is a reciprocal event, it could give rise to a second fusion gene, ABL-BCR, on the derivative 9q+. We analyzed the influence of the 3' M-BCR deletion on the clinical picture at diagnosis and disease outcome in 57 patients with a clinical diagnosis of CML. Molecular studies were done on DNA from peripheral blood leukocytes or bone marrow with the restrictions enzymes BglII, EcoRI, HindIII, and BamHI, and the BCR 3' probe (transprobe 1) (Oncogene Science Inc.), which encompasses almost all of the 5.8 Kb of the M-BCR gene area. In 18 patients Southern blot analysis showed deletion of the 3' end of BCR gene (32.7%). There were no significant differences between patients with or without deletion, either in the clinical and laboratory data at the disease diagnosis or at the disease outcome. The absence of differences between the patients with and without 3' BCR deletion supports the hypothesis that the hybrid gene ABL-BCR does not have an important role in leukemogenesis in CML cases.

Occurrence of BCR-ABL rearrangement in a Philadelphia chromosome-negative patient with 5q and 13q deletions and myeloproliferative syndrome.

We report the case of a patient with a myeloproliferative syndrome and traits of myelodysplasia and myelofibrosis whose karyotype showed 5q and 13q deletions, as well as Philadelphia chromosome negativity. A molecular biology study performed by Southern blot, with a probe covering the M-bcr region, led to detection of three bands other than the germinal ones, which hints at the possible existence of two cut points in the M-bcr region of an allele, or participation of both alleles. The patient presented a complex hematological picture, which might be explained on the basis of the cytogenetic and molecular findings.

Association of t(9;11)-MLL AF9 and trisomy 8 in an AML-M5 preceded by pancytopenia.

The implication of MLL gene rearrangements in the prognosis of acute myeloblastic leukemia is an issue of considerable current interest. We report a case of a young man who initially presented with a pancytopenia and went on to develop a highly-aggressive acute myeloblastic leukemia. At this time, the karyotypic study revealed trisomy 8, a t(9;11) was demonstrated by fluorescence in situ hybridization (FISH) and the MLL/AF4 rearrangement by reverse transcriptase-polymerase chain reaction (RT-PCR).

[The relationship between the persistence of the BCR/ABL gene and relapse in adult patients with acute lymphoblastic leukemia]. / Relación entre la persistencia del gen BCR/ABL y la recaída en pacientes adultos con leucemia linfoblástica aguda.

BACKGROUND: The Philadelphia chromosome (Ph') is originated by the t(9;22) which determines the rearrangement BCR/ABL. This rearrangement has been associated with an unfavourable prognosis in patients diagnosed with adult acute lymphoblastic leukaemia (ALL). PATIENTS AND METHODS: The BCR/ABL gene (p210 and p190) was prospectively studied by nested RT-PCR in 17 adult patients diagnosed with ALL BCR/ABL-positive cases were monitored by RT-PCR and cytogenetic techniques over the treatment period (LAL-93 AR protocol). RESULTS: BCR/ABL mRNA was detected in 8 out the 17 patients studied (47%). The Ph' chromosome was detected in 4 cases. Follow-up was completed in 6 out of the 8 BCR/ABL positive cases. PCR only became negative in one patient. The 5 patients with persistently positive BCR/ABL relapsed, whereas the case which became negative was still in complete remission after 24 months follow-up. In 3 out of the 4 Ph' positive patients, the karyotype was normal after induction therapy. CONCLUSIONS: This study clearly demonstrates the usefulness of molecular analysis in the diagnosis and follow-up of ALL compared with conventional cytogenetic techniques. The importance of molecular analysis to assess the efficacy of the treatment used has been emphasized and the poor evolution of BCR/ABL-positive patients has been confirmed.

Identification of a conserved erythroid specific domain of histone acetylation across the alpha-globin gene cluster.

We have analyzed the pattern of core histone acetylation across 250 kb of the telomeric region of the short arm of human chromosome 16. This gene-dense region, which includes the alpha-globin genes and their regulatory elements embedded within widely expressed genes, shows marked differences in histone acetylation between erythroid and non-erythroid cells. In non-erythroid cells, there was a uniform 2- to 3-fold enrichment of acetylated histones, compared with heterochromatin, across the entire region. In erythroid cells, an approximately 100-kb segment of chromatin encompassing the alpha genes and their remote major regulatory element was highly enriched in histone H4 acetylated at Lys-5. Other lysines in the N-terminal tail of histone H4 showed intermediate and variable levels of enrichment. Similar broad segments of erythroid-specific histone acetylation were found in the corresponding syntenic regions containing the mouse and chicken alpha-globin gene clusters. The borders of these regions of acetylation are located in similar positions in all three species, and a sharply defined 3' boundary coincides with the previously identified breakpoint in conserved synteny between these species. We have therefore demonstrated that an erythroid-specific domain of acetylation has been conserved across several species, encompassing not only the alpha-globin genes but also a neighboring widely expressed gene. These results contrast with those at other clusters and demonstrate that not all genes are organized into discrete regulatory domains.

The thalassemia syndromes: molecular characterization in the Spanish population.

This work compiles the results of our research on alpha- and beta-thalassemias, and includes a literature review of the molecular genetics of alpha- and beta-thalassemias in Spain. We studied 1,564 subjects with thalassemia (294 with beta-thalassemia and 1,264 with alpha-thalassemia) by molecular biology techniques. In relation to beta-thalassemia, a total of 15 different mutations were characterized in a study of 308 chromosomes belonging to 294 unrelated subjects. Eleven were homozygotes (22 alleles), three compound heterozygotes (6 alleles), and the remaining 280 were heterozygotes (280 alleles). A total of 86.6% of the alleles identified can be grouped into five different mutations [IVS-I-1 (G-->A), IVS-I-6 (T-->C), IVS-I-110 (G-->A), codon 39 (C-->T), codons 8/9 (+G)]. In 14 subjects (4.5%), all heterozygotes, it was not possible to identify the alteration responsible for the beta-thalassemia. For alpha-thalassemia, 911 subjects showed heterozygous alpha(+)-thalassemia (872 with -3.7 kb; 14 with -4.2 kb; two with the deletion of 3.5 kb of DNA, and 23 with nondeletional alpha-thalassemia). Two hundred and thirty-three subjects had homozygous alpha(+)-thalassemia (223 for -alpha(-3.7)/-alpha(-3.7)); one for -alpha(-4.2)/-alpha(-4.2); six for -alpha(-3.7)/-alpha(-4.2); one for -alpha(-3.5)/-alpha(-3.7); one for alphaalpha(Nco)/alphaalpha(Nco); one for alpha(HPh)/alpha(Hph)). One hundred patients presented with heterozygous alpha(0)-thalassemia (18 of whom were progenitors of patients with Hb H disease). The alpha(0) determinant was found in 20 patients with Hb H disease associated with -alpha(-3.7). From the DNA analysis were identified the - -(MED), - -(SEA), - -(SPAN) deletions and the - -(MA) mutations; in three cases, a break that affects the distal portion of the short arm of chromosome 16; one of these was associated with the ATR-16 (alpha-thal with mental retardation) syndrome. Triplication of the alpha genes (alphaalphaalpha(-3.7)/alphaalpha) was found in 25 subjects, 16 of whom were associated with a heterozygous beta-thalassemia. Only one patient was homozygous for the triplication of alpha genes (alphaalphaalpha(-3.7)/alphaalphaalpha(-3.7)) that was associated with a heterozygous beta-thalassemia. In the Mediterranean region preventive programs for thalassemia, based on the detection of heterozygote carriers and genetic advice, are not sufficient to reduce the incidence of newborns with major thalassemia. Prenatal diagnosis of thalassemias has given a new dimension to the prevention of these, but in order to implement this, a knowledge of the mutations and the incidence of these, is essential. This study, therefore, aims to give a general picture of the molecular genetics of thalassemia and its geographical distribution in our area.

[Value of analyzing reticulocyte subpopulations in polycythemia]. / Valor del análisis de las subpoblaciones reticulocitarias en el estudio de las poliglobulias.

OBJECTIVE: The evaluation of the RNA content in reticulocytes by means of flow cytometry allows us to differentiate three reticulocyte populations by their degree of maturity: LFR, MFR and HFR. We have studied the value of those reticulocyte subpopulations for the differential diagnosis in polycythemia. PATIENTS AND METHODS: We have studied 25 polycythemia patients, 12 of these were diagnosed of polycythemia vera (PV) and 13 of secondary polycythemia (PS). Reticulocytes and their different reticulocyte populations were automatically analyzed by flow cytometry using the Sysmex R-2000 analyzer (TOA Corp. Japan). We calculated the reticulocyte maturity index (RMI) which is the percentage of the equation (MFR + HFR) x 100/LFR (9). We have also studied hemogram parameters (Hct, Hb, VCM, HCM, RDW, leukocytes and platelets), iron metabolism (serum iron, ferritin, transferrin and transferrin saturation index), and erythropoietin levels. The findings were statistically analyzed using the Pearson correlation test and the comparison tests of averages of independent samples (Student's t). RESULTS: Patients with PV present high RMI and MFR + HFR (medium and high fluorescence reticulocytes) as compared with secondary polycythemia . We did not find any correlation between RMI and other analytical parameters (erythropoietin, ferritin, serum iron, transferrin), except for the transferrin saturation index (TSI), with a correlation factor r: 0.5486 for p < 0.05. CONCLUSION: This higher proportion of younger reticulocytes in PV might be explained because they are a parameter that would reflect the expansion of erythropoietic bone progenitor cells, which would be more increased in PV than in PS. However, another possible explanation would consist in the existence of associated ferropenia in PVs, which lead to an increase in cytoplasmic levels of transferrin receptor mRNA.

[Usefulness of molecular screening in childhood lymphoblastic leukemias]. / Utilidad de un screening molecular en las leucemias linfoblásticas pediátricas.

PURPOSE: To demonstrate that a molecular screening by reverse transcriptase polymerase chain reaction (RT-PCR) of TEL/AML1, E2A/PBX1 and BCR/ABL genes in pediatric acute lymphoblastic leukaemia is a rapid method that allows one to exceed the percentage of adult patients with the BCR/ABL rearrangement. PATIENTS AND METHODS: 12 Spanish children with acute lymphoblastic leukaemia were studied, 11 of them newly diagnosed and 1 relapsed. The patients were between 18 months and 10 years old. Bone marrow aspiration was collected between april and december 1996, RNA was isolated and cDNA was subjected to PCR amplification for TEL/AML1, E2A/PBX1 and BCR/ABL genes. Normal ABL and E2A genes were studied as amplification controls. RESULTS: One of these hybrid genes was found in 33.3% of patients studied. TEL/AML1 in two cases (16.6%), E2A/PBX1 in one case (8.3%) and BCR/ABL in another one (8.3%). CONCLUSIONS: On the basis of these data it would be useful to achieve a molecular screening of TEL/AML1, E2A/PBX1 and BCR/ABL genes in pediatric acute lymphoblastic leukaemia for allowing a molecular classification in a great percentage of patients that exceed the BCR/ABL positivity in adults.

Hb Johnstown [beta 109 (G11) Val-->Leu]: second case described and associated for the first time with beta(0)-thalassemia in two Spanish families.

Hb Johnstown, a high oxygen affinity hemoglobin, was identified in four members from two unrelated Spanish families with erythrocytosis and left-shifted hemoglobin-oxygen dissociation curve. This hemoglobin variant, electrophoretically silent, was analyzed by reverse-phase high-performance liquid chromatography, and the mutation was characterized at the DNA level by beta gene sequencing. In one of these families, two members are affected with Hb Johnstown in association with beta(0)-thalassemia. In these cases the erythrocytosis and low values for P(50) due to Hb Johnstown remain in spite of the beta-thalassemia.

Cleavage of the ALL1 gene in acute lymphoid leukemia before treatment disappears in relapse.

BACKGROUND AND OBJECTIVE: ALL1 gene rearrangements are frequently found in secondary acute leukemias (ALs). A site-specific cleavage of the ALL1 gene in a consensus sequence for topoisomerase II recognition has been considered to be the initial step leading to ALL1 rearrangement and subsequent therapy-related AL. The aim of the present study was to evaluate this cleavage in our patients, to analyze whether it is a laboratory-produced artefact and to check whether it persists or causes a real ALL1 gene rearrangement at relapse. DESIGN AND METHODS: We studied ALL1 rearrangement in 74 cases of AL before treatment by Southern blot avoiding room temperature exposure or delay in processing the samples which could produce ALL1 cleavage. DNA was available for two cases with ALL1 cleavage; it was analyzed by three different Southern blots in one and two in the other. One case with ALL1 cleavage was also studied in relapse. RESULTS: The presence of the cleavage of the ALL1 DNA was found in 3 of 74 (4%) patients. Two of these three patients had the ALL1 cleavage in three and two different analyses. One case was positive for ALL1 cleavage at diagnosis, but negative for both ALL1 cleavage and ALL1 rearrangement at relapse. INTERPRETATION AND CONCLUSIONS: The fact that a constant pattern was obtained from the same patients in different DNA preparations, supports the notion that ALL1 cleavage is not a laboratory artefact. The absence of the cleavage in a sample from a relapsed patient suggests that the subclone with the ALL1 cleavage, in this case, did not play a clear role in the pathogenesis of disease recurrence.

High incidence of the CD8/9 (+G) beta 0-thalassemia mutation in Spain.

BACKGROUND AND OBJECTIVE: In Spain, as in other Mediterranean regions the most common beta-thalassemia mutations are due to point mutations in gene regions that are critical for production of mRNA, such as [IVS-I-nt1 (G-->A), IVS-I-nt6 (T-->C), IVS-I-nt110 (G-->A)] which interrupt normal RNA processing or nonsense mutations [CD39 (C-->T)] which interrupt the translation of mRNA. The frameshift mutation CD8/9 (+G) is a very common allele in Asian Indians but is rare in the Mediterranean regions in which isolated alleles with this mutation have been found in Israel, Greece, Portugal and Turkey. DESIGN AND METHODS: We performed a molecular analysis of 175 chromosomes corresponding to 233 beta-thalassemia patients (221 heterozygous, 10 homozygous and 2 compound heterozygous) who belong to 169 Spanish families. The study of beta-thalassemia was made by PCR-ARMS, the alpha genes by Southern blot, the phenotype of Hb Lepore by enzymatic amplification and the presence of -158 gamma G C-->T mutation by PCR and digestion with the restriction enzyme XmnL. RESULTS: Twenty of these 233 patients showed the beta-thalassemia mutation CD8/9 (+G) (17 were heterozygous, 2 homozygous and in one patient the mutation was associated with a structural variant Hb Lepore Boston). INTERPRETATION AND CONCLUSIONS: These data reveal the heterogeneity of beta-thalassemia in Spain and the relatively high frequency (8.6%) of the frameshift mutation CD8/9 (+G). It is surprising that homozygotes for beta zero-thalassemia due to this mutation with very high Hb F values (around 90%) present a phenotype of intermediate thalassemia.

Detection of bcr/abl mRNA in a case of chronic myelogenous leukemia in long-term remission: CML or sensitivity of detection?

Chronic myelogenous leukemia (CML) is a myelo-proliferative disorder which, after a chronic phase which lasts an average of 3 years, evolves into an acute disease which is resistant to chemotherapy. Nevertheless, a few studies have reported cases in which partial or complete hematologic, cytogenetic and/or molecular remission of the disease were observed either spontaneously or after non intensive chemotherapy, with or without medullar aplasia. Some of these patients later relapsed into a blast crisis. We report a case of CML with clinical and hematologic remission for 19 years after two cycles of busulphan not causing medullar aplasia, negative for the BCR/ABL gene by Southern blot but with the gene's mRNA detectable by hot start nested RT-PCR.