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Determining the mechanisms by which genes are switched on and off during development and differentiation is a key aim of current biomedical research. Gene transcription has been widely observed to occur in a discontinuous fashion, with short bursts of activity interspersed with longer periods of inactivity. It is currently not known if or how this dynamic behaviour changes as mammalian cells differentiate. To investigate this, using a newly developed on-microscope analysis, we monitored mouse α-globin transcription in live cells throughout sequential stages of erythropoiesis. We find that changes in the overall levels of α-globin transcription are most closely associated with changes in the fraction of time a gene spends in the active transcriptional state. We identify differences in the patterns of transcriptional bursting throughout differentiation, with maximal transcriptional activity occurring in the mid-phase of differentiation. Early in differentiation, we observe increased fluctuation in the patterns of transcriptional activity whereas at the peak of gene expression, in early and intermediate erythroblasts, transcription appears to be relatively stable and efficient. Later during differentiation as α-globin expression declines, we again observed more variability in transcription within individual cells. We propose that the observed changes in transcriptional behaviour may reflect changes in the stability of enhancer-promoter interactions and the formation of active transcriptional compartments as gene expression is turned on and subsequently declines at sequential stages of differentiation.

In higher eukaryotes, many genes are regulated by enhancers that are 104–106 base pairs (bp) away from the promoter. Enhancers contain transcription-factor-binding sites (which are typically around 7–22 bp), and physical contact between the promoters and enhancers is thought to be required to modulate gene expression. Although chromatin architecture has been mapped extensively at resolutions of 1 kilobase and above; it has not been possible to define physical contacts at the scale of the proteins that determine gene expression. Here we define these interactions in detail using a chromosome conformation capture method (Micro-Capture-C) that enables the physical contacts between different classes of regulatory elements to be determined at base-pair resolution. We find that highly punctate contacts occur between enhancers, promoters and CCCTC-binding factor (CTCF) sites and we show that transcription factors have an important role in the maintenance of the contacts between enhancers and promoters. Our data show that interactions between CTCF sites are increased when active promoters and enhancers are located within the intervening chromatin. This supports a model in which chromatin loop extrusion1 is dependent on cohesin loading at active promoters and enhancers, which explains the formation of tissue-specific chromatin domains without changes in CTCF binding. Micro Capture-C allows physical contacts to be determined at base-pair resolution, revealing that transcription factors have an important role in the maintenance of the contacts between enhancers and promoters.

Mammalian genomes are subdivided into large (50-2000 kb) regions of chromatin referred to as Topologically Associating Domains (TADs or sub-TADs). Chromatin within an individual TAD contacts itself more frequently than with regions in surrounding TADs thereby directing enhancer-promoter interactions. In many cases, the borders of TADs are defined by convergently orientated boundary elements associated with CCCTC-binding factor (CTCF), which stabilises the cohesin complex on chromatin and prevents its translocation. This delimits chromatin loop extrusion which is thought to underlie the formation of TADs. However, not all CTCF-bound sites act as boundaries and, importantly, not all TADs are flanked by convergent CTCF sites. Here, we examined the CTCF binding sites within a ∼70 kb sub-TAD containing the duplicated mouse α-like globin genes and their five enhancers (5’-R1-R2-R3-Rm-R4-α1-α2-3’). The 5’ border of this sub-TAD is defined by a pair of CTCF sites. Surprisingly, we show that deletion of the CTCF binding sites within and downstream of the α-globin locus leaves the sub-TAD largely intact. The predominant 3’ border of the sub-TAD is defined by a steep reduction in contacts: this corresponds to the transcribed α2-globin gene rather than the CTCF sites at the 3’-end of the sub-TAD. Of interest, the almost identical α1- and α2-globin genes interact differently with the enhancers, resulting in preferential expression of the proximal α1-globin gene which behaves as a partial boundary between the enhancers and the distal α2-globin gene. Together, these observations provide direct evidence that actively transcribed genes can behave as boundary elements.Significance StatementMammalian genomes are complex, organised 3D structures, partitioned into Topologically Associating Domains (TADs): chromatin regions that preferentially self-interact. These chromatin interactions are thought to be driven by a mechanism that continuously extrudes chromatin loops, forming structures delimited by chromatin boundary elements and reflecting the activity of enhancers and promoters. Boundary elements bind architectural proteins such as CCCTC-binding factor (CTCF). Previously, an overlap between the functional roles of enhancers and promoters has been shown. However, whether there is overlap between enhancers/promoters and boundary elements is not known. Here, we show that actively transcribed genes can also behave as boundary elements, similar to CTCF boundaries. In both cases, multi-protein complexes bound to these regions may stall the process of chromatin loop extrusion.

We employ and extensively characterise an ex vivo culture system to study terminal erythroid maturation of CD34+ progenitors from the peripheral blood of normal individuals and patients with Congenital Dyserythropoietic Anaemia type 1 (CDA-I). Using morphological analysis, FACS analysis and the proteomic approach CyTOF, we analysed patient-derived erythroblasts stage-matched with those from healthy donors during the expansion phase and into early differentiation. In patient cells, aspects of disordered erythropoiesis manifest midway through differentiation, including increased proliferation and changes in the DNA accessibility profile. We also show that cultured erythroblasts from CDA-I patients recapitulate the pathognomic feature of this erythroid disorder with up to 40% of the cells having abnormal ‘spongy’ chromatin morphology by electron microscopy, as well as upregulation of GDF15, a marker of ineffective erythropoiesis. In the tertiary phase of culture, patient cells show significantly less enucleation and there is persistence of earlier erythroid precursors. Furthermore, the enucleation defect appears to be more severe in patients with mutations in C15orf41, as compared to the other known causative gene CDAN1, indicating a genotype/phenotype correlation in CDA-I. Such erythroblasts are a valuable resource for investigating the pathogenesis of this disease and provide the opportunity for streamlining diagnosis for CDA-I patients and ultimately other forms of unexplained anaemia.

β-Thalassaemia is one of the most common monogenic diseases with no effective cure in the majority of patients. Unbalanced production of α-globin in the presence of defective synthesis of β-globin is the primary mechanism for anaemia in β-thalassaemia. Clinical genetic data accumulated over three decades have clearly demonstrated that direct suppression of α-globin and induction of γ-globin are effective in reducing the globin chain imbalance in erythroid cells hence improving the clinical outcome of patients with β-thalassaemia. Here, we show that the histone deacetylase inhibitor drug, vorinostat, in addition to its beneficial effects for patients with β-thalassaemia through induction of γ-globin, has the potential to simultaneously suppress α-globin. We further show that vorinostat exhibits these synergistic beneficial effects in globin gene expression at nanomolar concentrations without perturbing erythroid expansion, viability, differentiation or the transcriptome. This new evidence will be helpful for the interpretation of existing clinical trials and future clinical studies that are directed towards finding a cure for β-thalassaemia using vorinostat.

In the era of genome-wide association studies (GWAS) and personalized medicine, predicting the impact of single nucleotide polymorphisms (SNPs) in regulatory elements is an important goal. Current approaches to determine the potential of regulatory SNPs depend on inadequate knowledge of cell-specific DNA binding motifs. Here, we present Sasquatch, a new computational approach that uses DNase footprint data to estimate and visualize the effects of noncoding variants on transcription factor binding. Sasquatch performs a comprehensive k-mer-based analysis of DNase footprints to determine any k-mer's potential for protein binding in a specific cell type and how this may be changed by sequence variants. Therefore, Sasquatch uses an unbiased approach, independent of known transcription factor binding sites and motifs. Sasquatch only requires a single DNase-seq data set per cell type, from any genotype, and produces consistent predictions from data generated by different experimental procedures and at different sequence depths. Here we demonstrate the effectiveness of Sasquatch using previously validated functional SNPs and benchmark its performance against existing approaches. Sasquatch is available as a versatile webtool incorporating publicly available data, including the human ENCODE collection. Thus, Sasquatch provides a powerful tool and repository for prioritizing likely regulatory SNPs in the noncoding genome.

To assess factors influencing the success of whole-genome sequencing for mainstream clinical diagnosis, we sequenced 217 individuals from 156 independent cases or families across a broad spectrum of disorders in whom previous screening had identified no pathogenic variants. We quantified the number of candidate variants identified using different strategies for variant calling, filtering, annotation and prioritization. We found that jointly calling variants across samples, filtering against both local and external databases, deploying multiple annotation tools and using familial transmission above biological plausibility contributed to accuracy. Overall, we identified disease-causing variants in 21% of cases, with the proportion increasing to 34% (23/68) for mendelian disorders and 57% (8/14) in family trios. We also discovered 32 potentially clinically actionable variants in 18 genes unrelated to the referral disorder, although only 4 were ultimately considered reportable. Our results demonstrate the value of genome sequencing for routine clinical diagnosis but also highlight many outstanding challenges.

PhenCode (Phenotypes for ENCODE; http://www.bx.psu.edu/phencode) is a collaborative, exploratory project to help understand phenotypes of human mutations in the context of sequence and functional data from genome projects. Currently, it connects human phenotype and clinical data in various locus-specific databases (LSDBs) with data on genome sequences, evolutionary history, and function from the ENCODE project and other resources in the UCSC Genome Browser. Initially, we focused on a few selected LSDBs covering genes encoding alpha- and beta-globins (HBA, HBB), phenylalanine hydroxylase (PAH), blood group antigens (various genes), androgen receptor (AR), cystic fibrosis transmembrane conductance regulator (CFTR), and Bruton's tyrosine kinase (BTK), but we plan to include additional loci of clinical importance, ultimately genomewide. We have also imported variant data and associated OMIM links from Swiss-Prot. Users can find interesting mutations in the UCSC Genome Browser (in a new Locus Variants track) and follow links back to the LSDBs for more detailed information. Alternatively, they can start with queries on mutations or phenotypes at an LSDB and then display the results at the Genome Browser to view complementary information such as functional data (e.g., chromatin modifications and protein binding from the ENCODE consortium), evolutionary constraint, regulatory potential, and/or any other tracks they choose. We present several examples illustrating the power of these connections for exploring phenotypes associated with functional elements, and for identifying genomic data that could help to explain clinical phenotypes.

Summary Accurate diagnosis of rare inherited anaemias is challenging, requiring a series of complex and expensive laboratory tests. Targeted next‐generation‐sequencing (NGS) has been used to investigate these disorders, but the selection of genes on individual panels has been narrow and the validation strategies used have fallen short of the standards required for clinical use. Clinical‐grade validation of negative results requires the test to distinguish between lack of adequate sequencing reads at the locations of known mutations and a real absence of mutations. To achieve a clinically‐reliable diagnostic test and minimize false‐negative results we developed an open‐source tool (CoverMi) to accurately determine base‐coverage and the ‘discoverability’ of known mutations for every sample. We validated our 33‐gene panel using Sanger sequencing and microarray. Our panel demonstrated 100% specificity and 99·7% sensitivity. We then analysed 57 clinical samples: molecular diagnoses were made in 22/57 (38·6%), corresponding to 32 mutations of which 16 were new. In all cases, accurate molecular diagnosis had a positive impact on clinical management. Using a validated NGS‐based platform for routine molecular diagnosis of previously undiagnosed congenital anaemias is feasible in a clinical diagnostic setting, improves precise diagnosis and enhances management and counselling of the patient and their family.

Two Spanish families with α thalassaemia, including 4 individuals with Hb H disease, are described. DNA mapping shows that, in addition to the common α thalassaemia determinant (-α3.7), a different and previously unreported allele is present in each family. In one, there is a deletion of 10.5–12 kb of DNA including both α genes (-SPAN). in the other, a deletion of more than 100 kb has removed the entire α globin gene complex (-BR).

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.

Hemoglobin switching is a complex process by which distinct globin chains are produced during stages of development. In an effort to characterize the process of hemoglobin switching in the zebrafish model system, we have isolated and characterized several embryonic globin genes. The embryonic and adult globin genes are found in clusters in a head-to-head configuration. One cluster of embryonic and adult genes is localized to linkage group 3, whereas another embryonic cluster is localized on linkage group 12. Several embryonic globin genes demonstrate an erythroid-specific pattern of expression early during embryogenesis and later are downregulated as definitive hematopoiesis occurs. We utilized electrospray mass spectroscopy to correlate globin genes and protein expression in developing embryonic red cells. The mutation, zinfandel, has a hypochromic microcytic anemia as an embryo, but later recovers in adulthood. The zinfandel gene maps to linkage group 3 near the major globin gene locus, strongly suggesting that zinfandel represents an embryonic globin defect. Our studies are the first to systematically evaluate the embryonic globins in the zebrafish and will ultimately be useful in evaluating zebrafish mutants with defects in hemoglobin production and switching. © 2003 Elsevier Science (USA). All rights reserved.

Current models suggest that tissue-specific genes are arranged in discrete, independently controlled segments of chromatin referred to as regulatory domains. Transition from a closed to open chromatin structure may be an important step in the regulation of gene expression. To determine whether the human alpha-globin cluster, like the beta-globin cluster, lies within a discrete, erythroid-specific domain, we have examined the long-range genomic organization and chromatin structure around this region. The alpha genes lie adjacent to at least four widely expressed genes. The major alpha-globin regulatory element lies 40 kb away from the cluster within an intron of one of these genes. Therefore, unlike the beta cluster, cis-acting sequences controlling alpha gene expression are dispersed within a region of chromatin that is open in both erythroid and nonerythroid cells. This implies a difference in the hierarchical control of alpha- and beta-globin expression.

The major positive regulatory activity of the human alpha-globin gene complex has been localized to an element associated with a strong erythroid-specific DNase I hypersensitive site (HS -40) located 40 kb upstream of the zeta 2-globin mRNA cap site. Footprint and gel shift analyses of the element have demonstrated the presence of four binding sites for the nuclear factor GATA-1 and two sites corresponding to the AP-1 consensus binding sequence. This region resembles one of the major elements of the beta-globin locus control region in its constitution and characteristics; this together with evidence from expression studies suggests that HS -40 is a primary element controlling alpha-globin gene expression.

ATRX syndrome is characterized by X-linked mental retardation associated with α-thalassemia. The gene mutated in this disease, ATRX , encodes a plant homeodomain-like finger and a SWI2/SNF2-like ATPase motif, both of which are often found in chromatin-remodeling enzymes, but ATRX has not been characterized biochemically. By immunoprecipitation from HeLa extract, we found that ATRX is in a complex with transcription cofactor Daxx. The following evidence supports that ATRX and Daxx are components of an ATP-dependent chromatin-remodeling complex: ( i ) Daxx and ATRX can be coimmunoisolated by antibodies specific for each protein; ( ii ) a proportion of Daxx cofractionates with ATRX as a complex of 1 MDa by gel-filtration analysis; ( iii ) in extract from cells of a patient with ATRX syndrome, the level of the Daxx–ATRX complex is correspondingly reduced; ( iv ) a proportion of ATRX and Daxx colocalize in promyelocytic leukemia nuclear bodies, with which Daxx had previously been located; and ( v ) the ATRX complex displays ATP-dependent activities that resemble those of other chromatin-remodeling complexes, including triple-helix DNA displacement and alteration of mononucleosome disruption patterns. But unlike the previously described SWI/SNF or NURD complexes, the ATRX complex does not randomize DNA phasing of the mononucleosomes, suggesting that it may remodel chromatin differently. Taken together, the results suggest that ATRX functions in conjunction with Daxx in a novel chromatin-remodeling complex. The defects in ATRX syndrome may result from inappropriate expression of genes controlled by this complex.

We studied the functional interaction between human embryonic zeta 2 globin promoter and the alpha globin regulatory element (HS-40) located 40 kb upstream of the zeta 2 globin gene. It was shown by transient expression assay that HS-40 behaved as an authentic enhancer for high-level zeta 2 globin promoter activity in K562 cells, an erythroid cell line of embryonic and/or fetal origin. Although sequences located between -559 and -88 of the zeta 2 globin gene were dispensable for its expression on enhancerless plasmids, they were required for the HS-40 enhancer-mediated activity of the zeta 2 globin promoter. Site-directed mutagenesis demonstrated that this HS-40 enhancer-zeta 2 globin promoter interaction is mediated by the two GATA-1 factor binding motifs located at -230 and -104, respectively. The functional domains of HS-40 were also mapped. Bal 31 deletion mapping data suggested that one GATA-1 motif, one GT motif, and two NF-E2/AP1 motifs together formed the functional core of HS-40 in the erythroid-specific activation of the zeta 2 globin promoter. Site-directed mutagenesis further demonstrated that the enhancer function of one of the two NF-E2/AP1 motifs of HS-40 is mediated through its binding to NF-E2 but not AP1 transcription factor. Finally, we did genomic footprinting of the HS-40 enhancer region in K562 cells, adult nucleated erythroblasts, and different nonerythroid cells. All sequence motifs within the functional core of HS-40, as mapped by transient expression analysis, appeared to bind a nuclear factor(s) in living K562 cells but not in nonerythroid cells. On the other hand, only one of the apparently nonfunctional sequence motifs was bound with factors in vivo. In comparison to K562, nucleated erythroblasts from adult human bone marrow exhibited a similar but nonidentical pattern of nuclear factor binding in vivo at the HS-40 region. These data suggest that transcriptional activation of human embryonic zeta 2 globin gene and the fetal/adult alpha globin genes is mediated by erythroid cell-specific and developmental stage-specific nuclear factor-DNA complexes which form at the enhancer (HS-40) and the globin promoters.

We have identified a remote, tissue-specific, positive regulatory element that is of major importance in determining the level of human alpha-globin gene expression. Stable transformants containing this DNA segment linked to the alpha gene in mouse erythroleukemia cells expressed human alpha mRNA at levels that are indistinguishable from those seen in interspecific hybrids containing the human alpha genes in their normal context on chromosome 16. Furthermore, all transgenic mice containing the alpha genes linked to this region expressed alpha-globin mRNA at high levels in erythroid tissues; and in one such mouse, readily detectable levels of human alpha-globin chains could be demonstrated in the peripheral blood. There is considerable similarity in the position, structure, and function of this region upstream of the alpha-globin complex with previously described elements within the beta-globin dominant control region (DCR). This is m marked contrast to other structural and functional differences between the two gene clusters. It seems likely that these critical, positive regulatory regions might provide target sequences through which coordinate regulation of the alpha- and beta-like globin genes is achieved.

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.

A combination of polymorphic DNA markers, cytogenetic analysis, and in situ hybridisation has been used for the high resolution assignment of the human alpha globin gene cluster on chromosome 16. Multiallelic DNA probes from within the alpha globin cluster were used to determine the number of copies of this locus in three cell lines containing trisomies of the short arm of chromosome 16 and one with a familial inversion, inv(16). The breakpoints in these rearrangements flank the alpha globin locus and locate a shortest region of overlap to 16p13.1. A meiotic crossover was also localised to this band. In situ hybridisation of biotinylated DNA probes to normal and inverted chromosomes 16 [inv(16)(p13.1;q22)] showed hybridisation sites at opposite ends of the chromosomes, consistent with this regional localisation.

We have characterised a cluster of CpG rich (HTF) islands in the alpha-globin complex and report here two unusual features: The human embryonic zeta 2-globin gene is associated with an HTF island within its 3' portion rather than at the 5' end. Furthermore at least two non-functional pseudogenes within the cluster (psi zeta 1 and psi alpha 2) are associated with CpG rich islands.

A series of restriction fragment length polymorphisms which are due to DNA rearrangements have been identified within two highly variable regions flanking the human alpha globin genes. The existence of such highly polymorphic areas provides a large number of individual genetic markers for the alpha globin gene cluster on chromosome 16. If, as seems likely, such regions occur frequently throughout the human genome they should be of considerable value in the antenatal diagnosis of genetic disease.

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.

(1982). Sickle Cell-Hemoglobin D Iran: A Benign Sickle Cell Syndrome. Hemoglobin: Vol. 6, No. 1, pp. 57-59.

The recently identified θ-globin gene subfamily conists of the θ1-globin gene located downstream from the α1 -globin gene1–5, and several other members including at least one truncated, processed pseudogene ψθ2 (refs 1,6). Unlike the θ1 -globin genes of the rabbit3 and galago4, the structure of these genes in the orang-utan and baboon1,5 and their flanking regions show no apparent defects that would prevent their expression. Both θ1 -globin genes are split into three exons with the potential to code for a polypeptide of length 141 amino acids. Besides differing by 26% in replace-ment-site substitutions, the θ1 and α1 -globin genes of the orang-utan and baboon also differ in their promoter structures, in the use of TGA versus TAA as the termination codon, and in the use of AGTAAA versus AATAAA as the polyadenylation signal1,5. In contrast, the two θ1,-globin genes from primates only differ by 1.7% in the replacement-site substitutions5. Here we present the complete DNA sequence of a cloned θ1 -globin gene of humans, and show that it contains no apparent defects that would abolish its expression. Furthermore, by primer extension of single-stranded oligonucleotide probes, we show that the θ1 -globin gene of humans is transcribed in an erythroleukemia cell line K562. Three messenger RNA species were detected, with 5′-ends mapping to ∼70 base pairs (bp) downstream from a TAT A promoter sequence, at 8 bp downstream from a GGGCGG promoter sequence and at 40 bp upstream from the ATG inititrion codon, respectively. Haemin treatment of the K562 cells slightly enhances the level of the longest θ1-transcript. Our results provide strong evidence that the θ1 -globin gene of humans is transcriptionally active in cells of erythroid orign, and suggests the presence of a functional θ1-polypeptide in specific cells, possibly those of early erythroid tissue.

The length polymorphism between the human zeta-globin gene and its pseudogene is caused by an allele-specific variation in the copy number of a tandemly repeating 36-base-pair sequence. This sequence is related to a tandemly repeated 14-base-pair sequence in the 5' flanking region of the human insulin gene, which is known to cause length polymorphism, and to a repetitive sequence in intervening sequence (IVS) 1 of the pseudo-zeta-globin gene. Evidence is presented that the latter is also of variable length, probably because of differences in the copy number of the tandem repeat. The homology between the three length polymorphisms may be an indication of the presence of a more widespread group of related sequences in the human genome, which might be useful for generalized linkage studies.

We have examined the molecular structure of the human alpha globin gene complex from individuals with a common form of alpha thalassaemia in which one of the duplicated pair of alpha genes (alpha alpha) has been deleted (-alpha 3-7). Restriction mapping and DNA sequence analysis of the mutants indicate that different -alpha 3.7 chromosomes are the result of at least three independent events. In each case the genetic crossover has occurred within a region of complete homology between the alpha 1 and alpha 2 genes. Since the -alpha chromosomes may reflect the processes of crossover fixation and gene conversion between the two genes, their structures may provide some insight into the mechanism by which the concerted evolution of the human alpha globin genes occurs.

The duplicated human embryonic alpha-like globin genes encode a 5' functional zeta (xi 2) gene and a highly homologous pseudogene (psi xi 1). We have identified chromosomes with a xi 2-xi 1 rather than a xi 2-psi xi 1 arrangement by genomic mapping and oligonucleotide analysis. The DNA sequence of a cloned downstream xi-like gene provides direct evidence for the conversion of a psi xi 1----xi 1 gene, by a xi 2 gene. We present data suggesting that this gene conversion, which removed the only identifiable inactivating mutation in the psi xi 1 gene, was an interchromosomal event. The xi 2-xi 1 arrangement is common in all eight populations studied representing a previously undescribed type of polymorphism between individuals. Stable mRNA transcripts from the converted gene are absent at 16-20 weeks of gestation when transcripts from the xi 2 gene are readily detectable.

Restriction endonuclease mapping of nondeletion alpha-thalassemia determinants from a variety of racial groups showed no detectable abnormalities within a 40-kilobase region of the zeta-alpha globin gene cluster. By using a zeta-specific probe, we defined three different types of interactions that give rise to Hb H disease, each involving a nondeletion alpha-thalassemia haplotype. mRNA analysis showed further diversity within these groups, indicating that there are at least three nondeletion determinations.

K562 human leukemia cells synthesize embryonic hemoglobins after culture in the presence of hemin. We have rigorously identified these hemoglobins by globin chain analysis and peptide mapping. No adult hemoglobin could be detected, and beta-globin synthesis was less than 2 ppm of total protein synthesis. Persistent embryonic globin gene expression is known to occur as a consequence of globin gene deletions. However, restriction endonuclease mapping showed that the globin gene complexes in K562 cells are indistinguishable from normal. Hemin increased the rate of embryonic globin synthesis. The pattern of hemoglobin synthesis proved to be stable when cells from different laboratories were compared. One line, however, synthesized large amounts of Hb X and very little Hb Portland in response to hemin. Hb X has been previously detected in human embryos; we show here that it has the composition epsilon 2 gamma 2 and is diagnostic of imbalanced chain synthesis or "zeta thalassemia." We have identified several agents that induce hemoglobin synthesis in K562 cells. Different inducers induced different patterns of embryonic hemoglobin synthesis but never any adult hemoglobin synthesis.

A new deletion causing alpha thalassemia has been characterised in a Greek family. Detailed mapping of the alpha gene complex shows that the deletion extends for 5.2 kb and removes the whole of the alpha 2 gene and the 5' end of the alpha 1 gene. The affected chromosome, therefore produces no normal alpha chains and results in a phenotype of alpha thalassemia 1.

The interaction of homozygous alpha thalassaemia 2 with homozygous sickle cell (SS) disease results in a generally milder haematological picture with less intravascular sickling, less haemolysis, and higher haemoglobin levels. Clinically, patients are generally more mildly affected, though not all vaso-occlusive complications are reduced. Thus there is a possibility that the advantages gained by inhibition of sickling have been offset by the rheological disadvantages of the higher haemoglobin level. The capillary bed in the perimacular region of the posterior pole has been used to examine the degree of vaso-occlusion in age and sex matched controls with SS disease with and without homozygous alpha thalassaemia 2. The results demonstrated significantly less capillary abnormalities in the perimacular region of patients with alpha thalassaemia, though the size of the foveal avascular zone and the grading of perimacular capillary drop-out did not differ between the 2 genotypes. These results are compatible with a mild inhibitory effect of alpha thalassaemia on vaso-occlusion of the macular vasculature in SS disease.

Rearrangements which are most readily explained by homologous crossover between misaligned segments of DNA in the region of the human embryonic zeta (zeta) globin genes have been identified in individuals of three different racial origins. These recombination events have resulted in a surprisingly high prevalence of chromosomes with single (0.4%) and triplicated (1.3%) zeta genes with apparently no significant effect on the phenotype.

The Sickled Cell: From Myths to Molecules. By Stuart J. Edelstein. Harvard University Press: 1986. Pp. 197. $25, £22.25.

Hemoglobin: Molecular, Genetic and Clinical Aspects. By H. Franklin Bunn and Bernard G. Forget. Saunders: 1986. Pp. 690. $85, £98.

Background: Mammalian transcriptomes contain thousands of long noncoding RNAs (lncRNAs). Some lncRNAs originate from intragenic enhancers which, when active, behave as alternative promoters producing transcripts that are processed using the canonical signals of their host gene. We have followed up this observation by analyzing intergenic lncRNAs to determine the extent to which they might also originate from intergenic enhancers. Results: We integrated high-resolution maps of transcriptional initiation and transcription to annotate a conservative set of intergenic lncRNAs expressed in mouse erythroblasts. We subclassified intergenic lncRNAs according to chromatin status at transcriptional initiation regions, defined by relative levels of histone H3K4 mono- and trimethylation. These transcripts are almost evenly divided between those arising from enhancer-associated (elncRNA) or promoter-associated (plncRNA) elements. These two classes of 5′ capped and polyadenylated RNA transcripts are indistinguishable with regard to their length, number of exons or transcriptional orientation relative to their closest neighboring gene. Nevertheless, elncRNAs are more tissue-restricted, less highly expressed and less well conserved during evolution. Of considerable interest, we found that expression of elncRNAs, but not plncRNAs, is associated with enhanced expression of neighboring protein-coding genes during erythropoiesis. Conclusions: We have determined globally the sites of initiation of intergenic lncRNAs in erythroid cells, allowing us to distinguish two similarly abundant classes of transcripts. Different correlations between the levels of elncRNAs, plncRNAs and expression of neighboring genes suggest that functional lncRNAs from the two classes may play contrasting roles in regulating the transcript abundance of local or distal loci.