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Introduction: In contrast to the classical thalassemias, two distinct thalassemias were recently described in which the molecular defect does not reside in the globin genes but in a transcriptional activator of alpha-globin genes. This protein, ATRX, is mutated in the rare inherited disease of alpha-thalassemias (AT) with mental retardation (ATR-X syndrome) whose affected individuals show a mild form of AT. In addition, and independent of the ATR-X syndrome, there have been approximately 100 case reports worldwide of the association of an acquired form of AT with hematological neoplasms, the large majority of those cases being MDS (ATMDS). The clinical characteristics of such patients encompass the typical features of the underlying hematological disorder plus microcytic anemia. The latter is due to massively reduced alpha-globin gene transcription resulting in excess hemoglobin H (HbH), as revealed by supravital staining of peripheral blood erythrocytes and hemoglobin electrophoresis. The molecular defect of ATMDS lies in a mutation of the ATRX protein and, thus, represents a form of acquired alpha-thalassemia. ATMDS shows a striking male preponderance for reasons that are as yet unclear. We systematically studied patients with MDS and low MCV and/or MCH but without iron deficiency for the possible presence of HbH cells (indicative of ATMDS). Supravital staining and sequence analysis of the ATRX gene were performed as described (Steensma et al. Blood.2004;103(6):2019–26). Results: Two female pts with AT-MDS were identified by this strategy. Pat 1 was a 69 year old pt who diagnosed with MDS of the subtype refractory cytopenia with multilineage dysplasia (RCMD), ringed sideroblasts and thrombocytosis (MDS/MPS overlap syndrome, JAK2 negative). She presented with microcytic anemia (Hb 7,5 g/dl, MCV 76,5 fl, MCH 22.4 pg). Supravital staining of a peripheral blood smear revealed massive erythrocytic HbH inclusions. Sequencing of the ATRX coding sequence revealed a novel missense mutation with an A>G transition in codon 2234. This mutation (D2234G) results in an amino acid substitution in ATRX exon 32. It represents the 14th ATRX mutation described thus far and, moreover, the first mutation detected in a female. Pat 2 (61 years old) with initially RCMD and microcytic anemia (Hb 10.4 g/ld, MCV 69 fl, MCH 15.2 pg) had increasing erythrocytosis of 6.93 Mio/μl maximum (also JAK2 negative). Molecular analysis of the ATRX gene also showed a not previously reported ATRX point mutation in exon 8 (G521A) which results in an amino acid change from cysteine to tyrosine and consequently in loss of a zinc finger. Conclusions: Though AT-MDS is mostly diagnosed in males we have identified two female patients, both showing novel ATRX somatic mutations. ATMDS might be more frequent in female than previously thought. Microcytic anemia in association with a hematological neoplasm, most commonly MDS, should alert to ATMDS which is easily diagnosed by supravital staining of peripheral blood smears. The remarkable thrombocytosis and erythrocytosis, respectively, in our 2 pts are at least suggestive of other phenotypic abnormalities possibly associated with the acquired ATRX genotypes on the MDS background.

The basic Helix-Loop-Helix (bHLH) transcription factor SCL is required for specification of haematopoietic stem cells (HSCs) and for differentiation of the megakaryocytic and erythroid lineages. bHLH proteins bind DNA as dimers and this has been thought to be a prerequisite for their function. We challenged this concept by showing that SCL DNA-binding activity was dispensable for some of its functions using SCL−/− ES cells rescued with a DNA-binding defective SCL mutant (SCL-RER) (Porcher et al. Development,1999). We have now studied the in vivo requirements for SCL DNA-binding activity in mice with a germ line SCL-RER mutation. In contrast to SCL knock-out embryos that die at E9.5 from absence of haematopoietic development, specification of primitive erythroid progenitors was observed in SCL RER/RER mutant yolk sacs. At day E12.5 and E13.5, homozygote mutant SCL RER/RER embryos were smaller and paler than wild-type (wt) and heterozygote littermates but presented at the expected mendelian frequency. Lethality was first observed at day E14.5. However, 7% of homozygote mice were born from heterozygous crosses. Surviving adult mice presented with mild microcytic hypochromic anemia. Assessment of progenitor replating potential showed qualitative defects with poor haemoglobinisation of homozygote-derived fetal and adult CFU-Es compared to controls. To understand the cause of the phenotype, expression levels of candidate target genes were assessed in fetal liver cells enriched for either early progenitors or late normoblasts. We observed decreased or increased mRNA expression of most genes tested in mutant-derived erythroid populations when compared to controls matched for differentiation stage, thereby showing that SCL DNA-binding activities are required for both activation and repression of target genes. As examples, mRNA for red cell membrane protein Band 4.2 was dramatically decreased; in contrast, alpha-globin expression was up-regulated in early progenitors, indicating that SCL DNA-binding activity might be required for repression of alpha-globin levels in this setting. We then pursued the analysis of SCL-mediated alpha-globin gene regulation by chromatin immunoprecipitation (ChIP) analysis and tested 4 DNase I hypersensitive sites (DHS) previously shown to bind SCL. From material derived from mutant fetal liver cells, we observed slight variations of SCL binding on 3 out of the 4 cis-acting elements when compared to controls. Importantly, there was a dramatic decrease in SCL binding on the fourth DHS site (HS-12). We concluded that SCL DNA-binding activity was likely to be directly required for repression of alpha-globin levels in erythroid progenitors. Interestingly, we have recently characterised the interaction of SCL with a co-repressor complex comprising the oncoprotein ETO-2 in erythroid cells. We have now shown by ChIP that ETO-2 occupies the alpha-globin locus on HS-12 in wt erythroid progenitors, but not in more mature cells, therefore suggesting a role for the SCL/ETO-2 complex in repression of erythroid-specific genes in the early stages of erythroid maturation. In conclusion, this in vivo model confirms the dispensability of SCL DNA-binding activity for specification of HSCs and allows characterisation of DNA-binding requirements throughout development. Combined with studies of the dynamic of SCL-containing multiprotein complexes during erythroid maturation, this model will help define the molecular pathways involved in erythropoiesis.

Although a great deal is known about the molecular mechanism underlying α thalassemia (thal), which is characterized by reduced or absent α-globin expression, the molecular basis of α thalassemia in Pacific Islanders is not fully understood. Previously single α gene deletion alleles (−α3.7 and −α4.2) were the only types of α thalassemia identified in this population, however these genotypes do not account for several cases of Hb H disease in the Melanesian Islanders of Papua New Guinea (PNG) and Vanuatu. To date, no mutations have been found in the α globin gene cluster of these individuals. To determine the cause of α thalassemia in this population, we studied 35 individuals from three unrelated families of Pacific Islanders. 16 of them had classical Hb H disease characterized by mild to moderate anemia (Hb; 10.3 ± 2.3 g/dL), microcytosis (MCV; 66 ± 3.5 fL), hypochromia (MCH; 19.8 ± 1.6 pg), positive Hb H by electrophoresis (5–18%) and numerous Hb H inclusion bodies in peripheral blood. Gene mapping revealed that six had normal genotypes (αα/αα) while the rest were heterozygotes for α+ thalassemia (−α/αα). Preliminary sequence analyses demonstrated that the α globin genes and their regulatory elements were entirely normal casting some doubt on whether this novel form of α thal is genuinely linked to chromosome 16. Using a combination of a long range haplotype analysis by MALDI-TOF to simultaneously genotype 80 SNPs covering 350 kb of the telomeric end of chromosome 16 (16p; 13.3) and several tandem repeat markers (VNTRs and HVRs), we demonstrated that the α thal phenotype is strongly associated with a single haplotype (IIIa;PNG) found in these cases. Consistent with the Hb H patients, individuals from these families and another 15 unrelated individuals (4.7%) identified in a population survey (n = 315) who were hypochromic (MCH < 25 pg), despite having normal α genes (αα/αα) and iron status, were also found to carry the same type of chromosome 16 linked to the affected haplotype. These findings indicate that this form of α thalassemia is unequivocally linked to chromosome 16. Subsequent linkage disequilibrium analysis (LD) in these cases limited the chromosomal interval containing the mutation to within 169 kb from the 16p-telomere. Extensive genomic mapping using more than 10 restriction enzymes and 37 plasmid and PCR probes together with Pulse Field Gel Electrophoresis (PFGE), covering 400 kb of 16p, excluded small deletions, insertions, rearrangements and chromosomal truncations in the affected segment of chromosome 16. Direct genomic sequencing of collectively 40 kb of the α globin genes, all known multispecies conserved sequences (MCS-R, 1–4) demonstrated no apparent mutation. Interestingly, interspecific somatic cell hybrid analysis of the affected chromosome 16 in a mouse erythroid leukemia (MEL) background did not recapitulate the phenotype of α thalassemia. These results suggest a novel mechanism, either at the genetic or epigenetic level, underlying the reduced expression of the α globin genes in the Pacific Islanders and understanding such mechanism might provide more insights on the complexity of stage- and tissue-specific gene regulation in human erythroid environment.

In a previous study, we described a form of nondeletion alpha- thalassemia (alpha T Saudi alpha) found in subjects of Saudi Arabian origin. In the current study, using synthetic oligoprobe hybridization and restriction enzyme analysis, we have demonstrated that the molecular basis of alpha T Saudi alpha is due solely to a single base mutation (AATAAA----AATAAG) in the polyadenylation signal of the alpha 2 gene and that the frameshift mutation in codon 14 of the linked alpha 1 gene is the result of a cloning artefact. The alpha 2 polyadenylation signal mutation occurs in other Middle Eastern and the Mediterranean populations and is responsible for the clinical phenotype of Hb H disease in some Saudi Arabian individuals with five alpha genes (alpha T Saudi alpha/(alpha alpha alpha)T Saudi). Evidence suggests that the (alpha alpha alpha)T Saudi haplotype has arisen as a result of a recombination between two misaligned chromosomes bearing the alpha T Saudi alpha defect.