Abstract
In almost all aspects of biomedical science, molecular pathology has brought unprecedented value in the diagnosis and management of human disease. Numerous commentators cite haematological disease as the leading genetic cause of global mortality and morbidity, and of these, those of the red blood cells are the most frequent. This narrative review, with a historical perspective, will discuss the role of genetics in these conditions, the leading pathology of red blood cells being the haemoglobinopathies, principally sickle cell disease and thalassaemia, with their many variants, and with potential roles for non-coding RNAs. The impact of genetics into conditions of the red cell cytoplasm will consider the enzymopathies, led by glucose-6-phosphate dehydrogenase deficiency, and extend to those of the cell membrane, causing disease such as hereditary elliptocytosis. Mutations in genes coding almost all the coagulation factors, and several platelet abnormalities, are discussed, as are those linked to conditions of iron overload. Previous, current and evolving technologies for diagnostic testing and their link with potential targeted therapeutic options for patient management are considered.
Introduction
The development of molecular genetics and its clinical consequence of molecular pathology has been revolutionary, and haematology has been to the fore in the development of these disciplines. For example, Friedrich Miescher is credited with the first description of DNA, obtained as nuclein, from the nuclei of leukocytes [], still the most useful source of genetic material. However, in the modern era, although several workers used the developing field of cytogenetics to investigate the aetiology of leukaemia [], the “breakthrough” report of molecular pathology in haematology can be precisely mapped to that of Nowell and Hungerford demonstrating the karyotypic abnormality that came to be known as the Philadelphia chromosome []. Whilst the hereditary nature of the haemoglobinopathies had long been established, and its protein basis elucidated in the 1960s, it was in the 1970’s that molecular genetics demonstrated the precise chromosome sites of abnormalities that caused the disease, and then later the exact nature of the lesion at the level of the nucleotide strand, work that led to gene therapy [–]. This review aims to describe both a historical perspective and the current leading advances in the molecular pathology of non-malignant haematological disease.
The Epidemiology of Haematological Disease
The Global Burden of Disease (GBD) study reported 55.9 million deaths in 2017, of which 834,700 were due to various forms of blood cancer and 104,600 due to haemoglobinopathies and haemolytic anaemias []. A separate report from 2016 reported a global prevalence of the major forms of blood cancer of almost 4 million people, a number dwarfed by the prevalence of all forms of haemoglobinopathy and haemolytic anaemia of around 2 billion people []. Thus, although the number of deaths due to blood cancer markedly exceeds that due to haemoglobinopathies and haemolytic anaemias by a factor of around 8, the prevalence of haemoglobinopathies and haemolytic anaemias exceeds that of blood cancer by some 500-fold.
These data, focussing on mortality, fails to address the morbid aspects of these conditions, which can be expressed as years lived with disability (YLD). The GBD report on this topic [] told of some 460,000 YLDs in 2016 due to the major forms of blood cancer, data which is also dwarfed by the 6,572 million YLDs linked to haemoglobinopathies and haemolytic anaemias, an increase of some 14-fold, although that the lower YLD of blood cancer may be due to its higher mortality. Indeed, in 2011, the World Health Organisation estimated that around 7% of the world’s population to be carriers of a potentially pathological haemoglobin (Hb) gene, whilst more recently it has been estimated that 3 million people are living with sickle cell disease (SCD) alone, with over 300,000 affected births each year [, ]. But just as there are numerous variants of blood cancer and haemoglobinopathy, there are also many other haematological diseases with a genetic component, several with an impact into other forms of anaemia and haemostasis.
Of the hundreds of non-malignant haematological diseases, with the exception of a few, hard data on mortality and morbidity are scarce. The GBD study reported deaths in 2017 due to the most common of these: 7,200 to the thalassaemias, 38,400 to sickle cell disorders, 16,700 to glucose-6-phosphate dehydrogenase (G6PD) deficiency, and 42,200 to other haemoglobinopathies and haemolytic anaemias []. G6PD deficiency was recognised as a leading public health issue in India in 1966 []. The GBD also provides data regarding the prevalence and YLDs of these conditions (Table 1), which point to great variation in the extent to which each of the conditions bring disability. For example, crudely dividing the YLDs by the prevalence of each condition brings figure of between 0.01 to 0.08 for the haemoglobinopathies (smaller in each of the traits), but of the order or 1 × 10−6 for G6PD deficiency and 7.8 × 10−5 for its trait. This may be interpreted as a reduced impact of G6PD in terms of YLDs per case and so point to the haemoglobinopathies bringing most of the burden of disease in this setting.
TABLE 1
| Condition | Prevalence | YLDs |
|---|---|---|
| Thalassaemia | 366,000 | 23,000 |
| Thalassaemia trait | 287,107,000 | 3,260,000 |
| Sickle cell disorders | 3,888,000 | 317,000 |
| Sickle cell trait | 461,124,000 | 1,555,000 |
| G6PD deficiency | 331,513,000 | 26,000 |
| G6PD trait | 865,927,000 | 1,000 |
| Other haemoglobinopathies and haemolytic anaemias | 61,625,000 | 1,370,000 |
Global prevalence and years lived with disability of the haemoglobinopathies and haemolytic anaemias.
G6PD, glucose-6-phosphatase dehydrogenase; YLD, years lived with disability. From references [, ].
Perhaps the first reports of molecular genetics to dissect the genome with a view to molecular pathology was in the haemoglobinopathies, with the development of techniques such as the generation of globin gene cDNA from reticulocyte mRNA, and its subsequent insertion into bacterial plasmids [–16]. The haemoglobinopathies may be classified with relative ease as the thalassaemias, the sickling disorders, and their hybrids.
The Thalassaemias
Molecular Genetics Unravelling the Basis of the Disease
During the 1960’s and early 1970’s, the use of Hb electrophoresis was the gold standard techniques for the study of abnormal globin chains, with advances in cell biology showing aberrant mRNA activity in thalassaemia. By mid-1970s the use of restriction nucleases and Southern blotting was consolidating the precise aetiology of the haemoglobinopathies at the level of the DNA [–16]. These advances included reports of undetectable α-globin mRNA and deletions in the gene for α-globin as the cause of α-thalassaemia, and a case report of -thalssaemia [17–19]. The latter is of historical value as it charts the use of specific techniques, such as the purification of reticulocyte mRNA by an oligo-thymine column, the use of this mRNA to generate cDNA using an RNA-dependent DNA-polymerase from the avian myeloblastosis virus, hybridisation of cDNA to ‘native’ DNA, and use of an in vitro cell-free translation model where globins are generated from purified mRNAs.
Of the several important proofs of concept were that, in contrast to normal mRNA, that from the affected patient generated α- and γ-globin but no β-globin, and their cDNA failed to anneal to normal β-globin DNA. A subsequent report identified three types of abnormal mRNA in beta°-thalassaemia–one with no detectable message, a second whose mRNA hybridised incompletely with cDNA, and a third that had intact mRNA that failed to translate, whilst another report pointed out that clinically severe and mild thalassaemias were linked to the deletion of both or a single α-globin gene, respectively [20, 21]. Orkin et al. used extensive restriction endonuclease digestion of DNA from patients with β°-thalassaemia, with fragments hybridised to β-globin cDNA, to demonstrate various deletions of the β-globin gene, some partial, some complete [22]. Other studies followed, showing a functioning ζ (zeta) gene in α-thalassaemia, and further studies of α-globin gene deletion [23, 24]. To these were added demonstration of the deletions of three of the four α-globin genes in HbH disease, and of all four genes in Hb Bart’s foetal hydrops, [25–27]. The latter, and others [28, 29] also examined the role of mRNA in these conditions, a process made relatively easy by harvesting mRNA from reticulocytes and other cells [–, 20]. Baralle and colleagues [30] used both Sanger and Maxam/Gilbert methods to sequence a 2,000-nucleotide section upstream of the ε-globin gene, a region now known to include a regulatory promotor region. Work of this nature was fundamental in that it subsequently pointed to a further genetic lesion, i.e., defects in a region outside an apparently normal functional gene (its promotor) could lead to a haemoglobinopathy [31].
A key point as regards many of these reports is that gene analysis was supported by protein electrophoresis, high-performance liquid chromatography (HPLC) and, in some cases, studies of the synthesis of globin chains, thus unequivocally defining the molecular basis of these diseases. A good example of this is the elucidation of the genetic basis of Hb Lepore, whose abnormal protein was demonstrated by starch block electrophoresis in 1958 [32], and whose genetic basis was confirmed 20 years later with the demonstration of a fusion of the β-globin gene and the δ-globin gene [33]. By the end of the 1970s, the mapping of the various isogenes of Hb, and their pathology, was becoming established and their locations defined: chromosome 16 for the α-globin gene locus (two copies: HBA1 and HBA2) and chromosome 11 for the β-globin gene (HBB) and the γ-globin gene locus [, 34–37], locations subsequently further defined as 16p13.3 and 11p15.4 respectively [38, 39].
A key methodological aspect of the work thus described was the used of section-specific restriction endonucleases to determine the presence or absence of particular sections of DNA and so produce a genetic map of a particular locus [40–42], but by the mid 1980s, nucleotide chain sequencing was becoming established. Sanger’s method was used to demonstrate the precise nucleotide sequence of a section of an intron in the β-globin gene of a G to T transversion [43], a T to G transversion [44] and an A to G transition [45], all causing a β-thalassemia. The Maxam and Gilbert method was used to report a case of α-thalassaemia with single base mutation in an α-globin gene at codon 116, that being GAG to UAG that formed a premature termination [46], whilst other part-sequenced the ε-globin gene [47]. We now describe these mutations as a single nucleotide polymorphism (SNP) [48].
A further initiative developed in the 1980s was the ability to use molecular genetics in the pre-natal diagnosis of hemoglobinopathies, initially using restriction endonucleases, then by lesion-specific oligonucleotide probes [49–54]. However, there remained a place for HPLC in determining the presence of abnormal Hbs in an antenatal setting [55–57]. At the end of the decade, Higgs and colleagues [58], and Cao and Murro [59], reviewed the genetics of the α-globin and β-globin gene loci respectively, summarising the various forms of abnormalities deletions, insertions, and SNPs causing different phenotypes of thalassaemia and other diseases of the α-gene and β-gene complex. Fuelled by work on transgenic mice, the 1990s saw molecular genetics used to identify the mechanism for the class switches of genes controlling the Hb isotypes from embryo to adult [60], the so-called “locus control region” (LCR). On the β-globin gene locus, this region is some 34 kB upstream (5′) of the ε gene [61], whilst others provided evidence of the involvement of the Erythroid Kruppel-like factor (subsequently re-named KLF-1, coded for by KLF at 19p13.13) in LRC regulation, binding a CACCC box in promotor regions, other important regions being a TATA box [62, 63]. A large deletion in a section of the LCR results in a form of gamma-delta-beta thalassaemia [64].
Next-Generation Sequencing (NGS)
As technologies developed in the 2000’s, principally those of NGS analysis [65, 66], additional insights into the genetics of the thalassaemias were reported. These included α-gene duplication, defects in β-thalassaemia, α-gene quadruplication, enhanced prenatal diagnosis of β-thalassaemia, and population screening for both forms of thalassaemia [67–70]. A further advance was the ability to detect foetal nucleic acid in maternal blood, a technique used, alongside amplification refractory mutation system (ARMS) analysis and pre-implantation genetic diagnosis [71–74]. In Sardina, with a population of 1.7 million people, the carrier rate for β-thalassaemia is around 12%, such that 1 in 250 newborns is affected by this disease. Methods such as ARMS and other platforms found 25% of foetuses to be normal, 50% to be healthy carriers and 25% to be affected by the disease: over 98% of the latter pregnancies were terminated [75].
The ability to identify and quantify copy number variation (CNV) by quantitative RT-PCR can be improved by droplet digital PCR [76, 77], whilst whole-exome sequencing can be used to identify aberrant gene activity in thalassaemia [78, 79]. Described in 2010, third generation sequencing includes methods focussing on single-molecule sequencing technologies [80–82]. Examples of these methods include the Pac-Bio method, nanoball technology, and the Oxford Nanopore system, the latter used to detect rare and complex thalassaemia variants, and α-thalassaemia in preimplantation genetic testing [83–87]. These technologies have the ability to overcome the current shortcomings of short-read NGS for exon-level CNV of the globin genes, and provide a single test solution for combined analysis of single nucleotide variants and CNV.
Gene Therapy for Thalassaemia
Whilst bone marrow transplantation is an established option for treatment, molecular pathology may provide the next step [, , 88, 89]. The discovery and development of molecular genetics that came to be known as clustered regularly interspaced short palindromic repeats (CRISPR) [90] that, linked with Cas-9 endonuclease [91], has promised much, winning the 2020 Nobel Prize for Doudna and Charpentier with hope for therapeutic genome engineering [92]. One of the first practical uses of this technology was to correct a mutated β-globin gene in an induced pluripotent stem cell line in vitro [93], a process repeated by many others, giving proof of concept, with Sanger sequencing showing a corrected HBB [94]. Originally reported in respect of lymphoid malignancies [95], a genome-wide association study of 4,305 Sardinians (a population at high risk of haemoglobinopathy) [96], showed that BCL11A (coded at 2p16.1) is strongly linked to levels of HbF [97], and that this was linked to SNPs in a 3 kB section of the second intron [98]. These and other data proposed the hypothesis that manipulation of this gene, normally minimally active in the healthy adult, may lead to an increase in red cell HbF, a desired treatment for many haemoglobinopathies [96–98].
The revolutionary paper supporting this hypothesis, reported a patient with transfusion-dependent β-thalassaemia, and another with sickle cell disease who were ‘transplanted’ with autologous CRISPR-Cas9–edited CD34+ stem cells that were engineered to reactivate the production of foetal Hb by targeting BCL11A [99]. Over a year later, Hb levels had risen from 90 to 141 g/L, and from 72 to 120 g/L respectively, with parallel improvements in quality of life. A similar clinical study reported editing the BCL11A enhancer to increase Hb levels from 82 to 150 g/L, and from 108 to 140 g/L in two patients with transfusion-dependent β-thalassaemia [100]. Although a successful strategy, a meta-analysis suggested that the promotor regions of gamma globin genes HBG1/2 may be a more efficient target than BCL11A [101]. Alternative uses of this technology include the silencing of genes for various red blood cell antigens, so creating red cells with considerable transfusion possibilities [102].
The Sickling Disorders
As indicated in Table 1, the prevalence of SCD exceeds that of thalassaemia over 10-fold, and that of sickle cell trait (SCT) over thalassaemia trait by 61% [, ]. Data from 2008 points to the domination of sickle disorders in the 340,000 births globally with leading the list of Hb disorders (Table 2) [103, 104], although by 2021 this figure had risen to 515,000, with 7.74 million people living with the disease [105]. The leading hypothesis, postulated 70 years ago, for this very high level of an apparently pathological mutation is that it provides protection from infection by Plasmodium falciparum [106, 107].
TABLE 2
| Condition | % |
|---|---|
| Sickle cell anaemia | 63.9 |
| Sickle cell disease | 16.1 |
| β-thalassaemia major | 6.8 |
| HbE β-thalassaemia | 5.6 |
| Sickle/β-thalassaemia | 3.3 |
| HbH disease | 2.8 |
| HB Bart hydrops (α°/α°) | 1.5 |
Breakdown of the proportions of births with a leading haemoglobinopathy.
Molecular Genetics and the Basis of SCD
The precise lesions in the Hb molecules in the sickling disorders has long been known, and the electrophoresis technique developed in the 1950s was extended to further refine knowledge of the amino acid nature of the sickling condition during the 1960s [108–110]. In the 1970s this was extended to the different globin species and their roles in various haemoglobinopathies [111], enabling putative maps of the globin amino-acid chains [112]. As with the thalassaemias [17–20], the 1970s also saw the genesis of the molecular pathology of sickling disorders, primarily with restriction endonucleases [113–115]. Perhaps the first major use of molecular genetics was made by Marotta and colleagues [116] in the definition of the lesion that causes SCD. They purified mRNA from sickle cell reticulocytes using an oligo(dt)-column, and from this used avian myeloblastosis virus RNA-dependent DNA polymerase to generate cDNA that was subsequently phosphorylated. Restriction endonucleases digested this cDNA, and fragments were sequenced by the method of Maxam and Gilbert, showing the HbS to be due to the result of a single nucleotide base mutation in the β-globin gene that converts the glutamic acid codon (GAG) at amino acid position 6 to one for valine (GTG) [117]. A further advance being the detection of β-globin fragments in cells from amniotic fluid that were consistent with sickle-cell trait [118, 119].
As the 1980s proceeded, the work with the use of restriction endonucleases regarding achieving gene maps of the α-globin and β-globin loci [33, 58, 120, 121] was being confirmed and extended by direct genetic analysis, with developments such as isotopic and non-isotopic probes, and their use (alongside PCR) in prenatal diagnosis [122–125]. Subsequent reports described in situ dot hybridisation, amplification by PCR of a 725 bp section of the β-globin gene with the sickle-cell anaemia point mutation in formalin-fixed bone marrow samples [126, 127], and allele-specific PCR, used to analyse the lesion in HbC [128, 129]. A 1992 review summarised the potential for using molecular genetics to insert functioning α-globin and β-globin genes into haemopoietic stem cells via a viral vector [130]. This period also saw the development of PCR heteroduplex analysis, used to screen for HbS and HbC, and the use of amplification refractory mutation system to detect HbA/HbS chimerism after bone marrow transplantation for sickle cell anaemia [131–133].
Molecular Genetics Meets Cell Biology
Developments in cell biology and transfection were used in numerous studies of the basic biology of the sickle phenotype and genotype, with prototype gene therapy. Tang et al. [134] showed that the promoter sequences of the δ-globin gene differs from the β-globin gene by the absence of an erythroid Krüppel-like factor (EKLF) binding site and alteration of the CCAAT box to CCACC, and that restoration of this site can increase δ-globin gene expression, a strategy that has potential future clinical benefit. Knowledge of the sequence of the promotor region of the γ-globin gene enabled Graslund and colleagues [135] to insert an artificial transcription factor for this gene into a cell line, which resulted in an increased expression of α2γ2 HbF, and so a strategy for increasing levels of this Hb variant in SCD, a leading therapeutic option [136]. Ho and colleagues [137] generated recombinant mutants of sickle Hb in E coli to show that the formation of HbS polymerisation requires interaction between α-globin 114Pro and β-globin 87THr, and that a mutant 114Arg inhibits the polymerisation, whilst Cole-Strauss et al. [138] inserted a chimeric DNA/RNA to lymphoblastoid cells in a direct correction of the mutation in the Hb betaS allele.
A step closer to clinical application was taken by Pawlik and colleagues [139], who corrected SCD in a mouse model by inserting a lentivirus vector carrying a beta globin gene variant that prevents HbS polymerisation into haemopoietic stems cells, which was then passed into the mouse. They subsequently transduced human cord blood cells with the vector, and transplanted them into mice, using PCR to demonstrate integration of the human gene, with increased levels of the modified β-globin [140, 141]. Small-interfering RNA represents a new opportunity to treat various diseases [142], an example being use of a small interfering antisense RNA to silence a βs-globin gene when transfected into HeLa cells and an erythroleukaemic cell line [143]. Vasavda and colleagues [144] used RTqPCT to measure circulating HBB DNA in the plasma of patients with SCD, HbS/βo thalassaemia, HbSC and HbAA controls, finding no difference in steady state disease, but increased levels (alongside CRP and the white blood cell count) when in crisis.
A long-addressed strategy in SCD is to increase levels of HbF, and so ameliorate the clinical effects of the mutation, as does hydroxyurea, a drug that reduces levels of plasma HBB [145]. Using a variety of techniques that included mass spectrometry, RT- PCR and a custom-made Illumina platform, Sebastiani and colleagues [146] reported links between HbF and SNPs in TOX at 8q21.1, in GPM6B at Xp22.2, and at 15q22 (a region that includes AQP9, MAP2K1, SMAD3, and SMAD6), suggesting manipulation of these gene may influence levels of HbF. As in thalassaemia [99–101], BCL11A may be important in SCD. Zhou et al. [147] showed that knockdown of KLF1 in adult erythroid stem cells markedly reduces BLC11A levels and increases the ratio of γ-globin to β-globin. Uda et al. [96] used genome-wide association studies to show that BCL11A is associated with HbF levels in sickle cell patients (as it is in β-thalassaemia), whilst Ghedira and colleagues [148] used quantitative multiplex PCR to estimate that the loss of the BCL11A binding domain leads to an increase in HbF of 27 g/L. Chen and colleagues [149] used an electrophoretic mobility shift assay and other methods to show links between a SNP in a GATA-1 binding motif and high levels of HbF. Other genes linked to HbF include OR51B5 and OR51B6, both coded at 11p15.4, HBS1L-MYB at 6q23, and SAR1A at 10q22.1 [150–152].
Gene Therapy for SCD
Both these strategies came to clinical fruition in the decade that followed, fuelled by the slow and steady increase in knowledge of practicalities of gene therapy, such as inserting genes into cells [153–160] (Table 3). A comparative study concluded that BCL11A is the most clinically relevant approach for CRISR/Cas9 insertions into stem cells [161]. In 2017, Ribeil and colleagues [162] published a case report of a thirteen-year-old boy with homozygous SCD who was transplanted with his own CD34+ stem cells that had been engineered with a Lentivirus vector to carry the HBB variant βA-T87Q, a molecule that inhibits the sickling process. He was discharged on day 50, and after 15 months, the patient’s total Hb increased from 101 to 118 g/L with a fall in reticulocytes from 238,000 to 141,000 per mm3, with no recurrence of pathological sickle crises. In 2021, Esrick and colleagues [163] reported six patients with SCD who were transplanted with autologous CD34+ stem cells transduced with a lentivirus vector carrying a short-hairpin RNA targeting BCL11A. HbF induction was robust and stable, with all indices of HbF improved (e.g., HbF rising from 9.0 to 18.6 pg/cell) with a reduction or absence of clinical manifestation of the disease.
TABLE 3
| Method | Example |
|---|---|
| Lentivirus vector | An engineered HBB designed to impede red cell sickling into human CD34+ bone marrow cells, the resulting red cells exhibiting less sickling upon deoxygenation [153] |
| Lentivirus vector | Use of a small hairpin RNA to knockdown BCL11A in normal and sickle cell CD34+ cells generating red cells with γ-chain expression and so increased HbF [154] |
| Sleeping beauty transposon | An anti-sickling gene into CD34+ from a sickle cell patient which produced red cells with a less pathological phenotype [155] |
| Tal-effector nuclease | A full-length β-globin gene inserted into K562 (a myeloid leukaemia cell line) [156] |
| CRISPR/Cas9 | Correction of SCD patients bone marrow CD34+ cells and production of wild-type haemoglobin A [157] |
| CRISPR/Cas9 | Deletion of a 13 kB section of the β-globin locus in normal CD34+ stem cells, thus mimicking HPFH, resulting in increased expression of γ-globin gene expression [158,159] |
| CRISPR/Cas9 and adenovirus vector | Correction of the mutation responsible for SCD in patient-derived stem and progenitor cells with expression of adult β-globin (HbA) messenger RNA in erythrocytes [160] |
Methods for introducing genes into cells.
Figures in parentheses are references.
As descrived previously, the same transplant strategy was used by Frangoul et al. [99] to treat a 19-year-old female with the βo/β+ thalassaemia but also a 33-year-old female with SCD (βS/βS and a single α-globin deletion). This resulted in increases in total Hb from 90 g/L to 142 g/L after 15 months, and from 72 to 120 g/L, whilst the % of red cell expressing HbF increased from 10.1 to 100, and from 33.9% to 98.1%, respectively. Vaso-occlusive events were eliminated in the patient with SCD. The following year, Kanter and colleagues [164] reported the use of a lentivirus vector to insert a gene coding an antisickling engineered variant of HbA into autologous stem cells, subsequently transplanted back into the patient with SCD. The largest study so far (n = 35) of this engineered Hb saw total Hb rise from a median of 85 g/L to over 120 g/L (53% being the engineered Hb) after 36 months, with no severe vaso-occlusive events after transplantation. Levels of serum lactate dehydrogenase and serum indirect bilirubin, and the reticulocyte count, all fell markedly. Magrin and colleagues [165] reported a 36, 42, and 60 months follow up of three SCD patients with the antisickling Hb variant βA-T87Q, noting marked variation in the % of total Hb being composed of HbF and HbAA-T87Q of 43.7%, 14.0% and 51.1% respectively. Sharma et al. infused stem cells bearing a CRISPR-Cas9 product engineered to disrupt HBG1 and HBG2 into three patients with severe SCD [166]. This resulted in sustained induction of red cell foetal Hb, that being 19%–26.8% of total Hb, with 69.7%–87.8% of erythrocytes classified as F cells, and a decrease in the manifestations of SCD. Extending their previous work [163], Esrick and colleagues now [167] compared their patients transplanted with BCL11A-silenced stem cells with transplanted patients being treated with standard care hydroxyurea. The % of red cells from transplanted patients containing ≥10 pg of HbF was comparable to that of hydroxyurea high-responders, but superior to that of hydroxyurea low-responders, but the % of cells carrying HbS was lower than both hydroxyurea groups.
Whilst gene therapy represents an alternative to bone marrow transplantation, which although well-established, has its drawbacks [168], the number of treated patients is small and follow-up short, such that full assurance is as yet unforthcoming. Indeed, despite the apparent success of some cases where all three patients in the OTq923 study (targeting BCL11A) [166] showed a clinical improvement, they continued to have mild haemolysis and some symptoms of sickle-cell disease, a finding which may have contributed to the decision by the sponsor to discontinue the development of this product [169]. The treatment cost of $2 million may be balanced by reduced cost of, for example, vaso-occlusive events, acute chest pathology etc. over the hoped-for decades of life remaining and the inestimable cost of improved quality of life [170]. Chapman et al. have pointed out the danger of vector insertion related leukaemias in other diseases from 2003 to 2014 and found an increased frequency of potential driver mutations associated with myeloid neoplasms or clonal haematopoiesis in both genetically modified and unmodified stems cells after SCD transplantation [171]. It remains to be seen if these mutations precipitate other disease and prompts the possible need to screen for these mutations before gene therapy is commenced.
Concurrent Thalassaemia and Sickling Disorders
Electrophoresis and other methods have demonstrated the co-inheritance of different variants of the hemoglobinopathies, largely concurrent thalassaemia and SCD, but also of HbC with α-thalassaemia, and HbC with HPFH ([172–176]). These have subsequently been confirmed by techniques such as allele-specific PCR and restriction endonuclease mapping [177–180]. Kundrapu and colleagues used Sanger sequencing to define a compound heterogeneity in Hb D-Ibadan and HbC [181], whilst Wilcox and colleagues confirmed the genetic basis of Hb Kenya as being due to non-homologous crossing-over of β-globin and γ-globin genes, resulting in a fusion protein [182], and Redding-Lallinger et al. used NGS to define the gene lesions in HbS/HbD-Ibadan and β+thalassaemia/Hb D-Ibadan [183]. Notably, the genetics distinguished the Ibadan variant of HbD from the Los Angles variant, a feat not possible with electrophoresis.
Non-Coding RNAs (ncRNAs) in the Haemoglobinopathies
Whilst ncRNAs include ribosomal and transfer RNAs, these molecules are taken to include two major groups, the long non-coding RNAs and the microRNAs known to have regulatory roles in a number of diseases [184]. One of the first examples in the haemoglobinopathy setting involved insertion of a lentivirus vector carrying a small nuclear RNA into stem cells from a patient with β-thalassaemia, resulting in the correction of an aberrant splice site and increased levels of HbA [185]. Chen and colleagues, using RT-PCR and micro-array analysis, reported a large number of miRNAs in reticulocytes and red cells: mean miRNA expression was markedly higher in HbSS reticulocytes, whilst miR-320 expression was markedly higher in HbAA erythrocytes, whilst low levels in HbSS cells were linked to defective CD71 downregulation [186]. Notably, in view of the interest in BCL11A as a target for gene therapy [96–101, 161, 162, 166], miR-486-3p and miR-210 are regulators of this gene, with over-expression resulting in BCL11A suppression and so increased expression of the γ-globin gene [187, 188]. Others used RT-PCR to determine plasma levels of miR-451 and miR-155, finding increased expression in β°-thalassaemia/HbE, and a link between miR-451 in severe disease and reticulocyte counts [189]. Lai et al. [190] used a reticulocyte RNA microarray analysis in patients with HPFH and β-thalassaemia, reporting that 862 lncRNAs (605 upregulated, 257 downregulated) and 568 mRNAs (324 upregulated, 244 downregulated) showed a >2-fold change. Although much development work is needed, several commentators have hypothesised uses for ncRNAs as therapeutic options [191, 192].
The Enzymopathies
Whilst there are numerous mutations in genes coding for enzymes and other forms of haemolytic anaemia (Table 1), such as those linked to abnormal components of the erythrocyte membrane, the leading non-haemoglobinopathy genetic disease is glucose-6-phosphate dehydrogenase (G6PD) deficiency [, , ]. This disease is often cited as the most common enzymopathy, which in 1996 affected some 400 million people globally [193], and 20 years later had risen to almost 1.2 billion [, ]. Despite this figure, the number of deaths due to this disease is roughly twice that of the thalassaemias, but half that of the sickling conditions, due mainly to the more benign course of the disease and the variability and penetrance of the many mutations of its associated gene at Xq28.
As with the haemoglobinopathies, the leading driver of the deficiency is protection from malaria [194, 195]. A report from India in 1966 indicated a biochemically defined frequency of 3.6% in males and 7.7% in females in a population from Bengal, 7.4% in males and 31.0% in females in a population from Bombay, and 4.6% in a population from Calcutta unstratified for sex []. A later review described a global prevalence ranging from 2.9% in the Pacific region, 3.9% in Europe, to 6.0% in Africa, although these data depend on the method of assessment, which ranges from 3.6% for the NADPH fluorescence method to 6% for DNA analysis [196]. In Arab countries, prevalence ranges from 2% to 31% [197], and in countries targeting malaria elimination, an allele frequency of 5.3% has been reported [198].
From a partial amino acid sequence of the enzyme, Persico et al. synthesised a 17-mer probe from which to screen a cDNA library prepared from partially purified G6PD mRNA, with sections sequenced by the methods of Maxam and Gilbert, and of Sanger [199]. From this, the same group published further details of the 18 kB gene and its 13 exons and subsequently used Sanger sequencing of λ-phage cDNA clones to report SNPs in seven variants whose red cell enzyme activity ranged from <2% to 84% of normal levels [200]. Concurrently, Takizawa and colleagues purified G6PD to homogeneity, derived its 531 amino acid sequence, and from this constructed a 41-mer synthetic sequence subsequently labelled with P32 [201]. They then constructed bacteriophage vector cDNA libraries using mRNA from hepatocytes and a hepatoma cell line, which they transfected into E coli, screened selected colonies by hybridising with the 41-mer probe, and sequenced selected fragments by Sanger’s method. Vulliamy et al. [202] used a similar phage vector library strategy to demonstrate SNPs in G6PD from patients with G6PD deficiency that would lead to an abnormal product and so the haemolytic anaemia typical of the disease. Thus, whilst the molecular pathology of G6PD deficiency lags somewhat behind that of the haemoglobinopathies, the genetic basis of the disease has been elucidated [193, 203].
The second most important enzymopathy, the autosomal recessive pyruvate kinase deficiency (PKD), is caused by loss-of-function mutations in the coding gene PKLR at 1q22, and which is used in diagnosis and population screening [204]. With a frequency of some 1/20,000, the >300 mutations lead to viable clinical pictures, and overall PKD brings a 10-year reduction in lifespan with a hazard ratio (95% confidence interval) of 5.0 (1.9–13.4) [205]. Although there are examples of successful bone marrow transplantation [206], molecular therapies are as yet unreported.
Haemostasis
This section is easily divisible into pathology of the platelet and of coagulation factors, the latter being dominated in a clinical setting by haemophilia.
Haemophilia
Perhaps the oldest known genetic disease, as described in ancient Hebrew texts, haemophilia is the likely reason for the dispensation of the surgical removal of the foreskin (circumcision) in a neonate if older brothers died following prolonged bleeding after their own circumcision. In the modern era, the disease was clearly described over 200 years ago [207], although not named, and perhaps the oldest detailed clinical description of haemophilia is over 150 years ago [208], with a treatment of the infusion of serum at the beginning of the last century [209].
The gene for Factor VIII (F8) was cloned, sequenced (reporting 186 kB, 25 introns, and 26 exons) and its 2,351 amino acids (giving a mass of 267-kDa) determined in 1984 [210–213]. Shortly afterwards, FVIII-specific probes were used for the prenatal diagnosis of haemophilia, and its chromosomal location determined [214–217]. By the end of the decade, deletions, SNPs, inversions, and insertions in the gene had all been shown to be the cause of haemophilia [218–222], reviewed in [223]. Interestingly, a cluster of mutations, many of which are inversions, in intron 22 found in 40% of one series of haemophiliacs can cause defective joining of exons 22 and 23 in the mRNA and so result in the disease [224].
The closely-related Christmas disease was described in 1952 [225], the identity of the restorative agent (anti-haemophilic factor) was determined in the late 1950s as Factor VIII. Becoming known as haemophilia B to distinguish it from ‘standard’ haemophilia, subsequently known as haemophilia A, the gene coding Factor IX (i.e., FIX) was cloned in 1982, defects shown in 1985 to be the cause of deficiency of the protein [226–228]. Kurachi and Davie [229] used a baboon liver mRNA to synthesise a radiolabelled cDNA 14-mer primer to identify and clone sections of a human liver cDNA library that were inserted into plasmids. Restriction endonuclease digestion of positive clones provided fragments that were sequenced by the method of Maxam and Gilbert. The gene has eight exons transcribing a 2.8 kB RNA that translates to a pre- and pro-peptide and mature protein of 415 amino acids, subsequently cleaved to FIXa with a relative molecular mass of 57 kDa. Standard sequencing reported 55.8% of 1692 abnormalities to be point mutations, 20.8% to be polymorphisms,16.6% to be deletions, and 83.9% to be in exons, the most common being in the eighth (37%) and second (12%) [230]. The power of NGS was demonstrated by the analysis of DNA from 2401 patients with haemophilia A and 599 with haemophilia B, finding 924 unique variants, confirming the inversion in intron 22 to be the leading mutation in severe cases of haemophilia A (42% of cases), followed by frameshift and missense (both 17.4%), whilst missense mutation were the most common in mild and moderate disease (79.5%) [231]. In haemophilia B, the leading mutation was missense in both severe (47.2%) and mild/moderate (87.1%) disease.
The relatively small size of F9 (∼34 kb), normally coding a 2.8 kb mRNA, permits its insertion into a viral vector that can be delivered to hepatocytes that then produce the active zymogen product of some 461 amino acid residues [232]. Furthermore, molecular genetics has provided a further advance in identifying a mutated form (FIX-Padua) that generates a F9 with enhanced enzyme activity compared to normal F9, and which is effective in a clinical setting [233]. This part-contrast with F8, of 186 kb, coding an mRNA of 9 kb and ultimately a 2,300 amino acid product, that together provided a challenge for genetic engineers [234]. However, these have been overcome, and two therapeutics (Valoctocogene Roxaparvovec and Efanesoctocog Alfa) are now available for the treatment of haemophilia A [235].
Other Coagulation Factors
By 1971 the hereditary nature of dysfibrinogenaemia (as autosomal dominant), afibrinogenemia, hypoprothrombinaemia, dysprothrombinaemia, defects of Stuart-Prower factor (Factor X) (all autosomal recessive) had been described, all pointing to a genetic lesion [236]. The molecular pathology of all other factor deficiencies was subsequently defined, locations of leading coagulation genes, and their population frequencies, are shown in Table 4.
TABLE 4
| Gene | Location | Frequency of deficiency per million |
|---|---|---|
| F2 | 11p11.2 | 0.5 |
| F5 | 1q24.2 | 1 |
| F7 | 13q34 | 2 |
| F8 | Xq28 | 200a |
| F9 | Xq27.1 | 33a |
| F10 | 13q34 | 1 |
| F11 | 4q35.2 | 1 |
| F12 | 5q35.3 | 1 |
| F13 | 6p25.1 and 1q31.3 | 0.5 |
| vWf | 12p13.31 | 1b |
Coagulation factor genes.
Males.
The most severe phenotype. Factor XIII is composed to two protein chains.
Based on an amino acid sequence of a factor Va light chain, Jenny and colleagues [237] synthesised a 39-mer probe and used it to screen an oligo(dT)-primed human foetal liver library, with clones expressed in phage vectors. Overlapping fragments were sequenced by Sanger technology, reporting a 6672 bp coding region ultimately producing a mature protein of 330 kDa consisting of 2196 amino acids. Subsequent analysis showed the presence of 25 exons, and that removal of a domain from the protein structure generates FVa, formed from a 105 kDa heavy chain and a 71 or 74 kDa light chain [238]. The combined deficiency of FV and FVIII may follow loss-of-function mutations (mostly insertion/deletions and splice site mutations) in LMAN1 at 18q21.32, and MCFD2 at 2p21 that together code for membrane proteins of the endoplasmic reticulum and Golgi apparatus pathway for transporting the coagulation factors [239].
The gene for FVII is located 2.8 kB telomerically to that for FX, spanning 12 kB and with nine exons coding for a mature protein of 406 amino acids of 50 kDa, the leading cause of deficiency being a 10 bp insertion polymorphism, with other abnormalities linked to a variable reduction in biological activity [240]. Fibrinogen is formed from three proteins, coded for by FGA (7.6 kB, 6 exons), FGB (8 kB, 8 exons), both at 4q31.3, and FBG (8.5 kB, 10 exons) at 4q32.1, giving a total mass of 340 kDa: the estimated prevalence of deficiency (generally a deletion, most often in FGA) is around 1/million. Vitamin K dependent coagulation factor deficiency, producing low levels of several of the products of these genes, is linked to GGXC at 2p11.2, coding for gamma-glutamyl carboxylase, and VKORC1 at 16p11.2, coding for a subunit of the vitamin K epoxide reductase complex [241–243].
The most common genetic haemorrhagic disease is von Willebrand disease (VWD), with an estimated Caucasian population frequency of 0.57%–1.15%, translating to 5,700 to 11,500 per million [244], far exceeding that of haemophilia A at 200 per million males. However, clinical VWD runs a spectrum from asymptomatic to life-threatening, where the phenotype resembles haemophilia, such that the referral rate for the most severe cases has been estimated at 23 to 113 per million [245]. The variable nature of the disease, characterised by low levels of von Willebrand factor (vWf), can be accounted for by various mutations in VWF at 12p13.31, where it spans 176 kB, consisting of 52 exons between 52 base pairs and 1.3 kB generating an mRNA around 9 kB long [246, 247].
In contrast to low levels of plasma vWf, increased levels are common in cardiovascular disease, cancer, and in connective tissue diseases, reflecting an endothelial cell pathology, although the driver(s) are unlikely to be genetic [248]. Nevertheless, a leading feature of this molecule is as a chaperone for FVIII, increased levels of which are a risk for a venous thrombosis, with both molecules considered by some to be part of a thrombophilia screening panel [249], although this view is not universal [250]. Genes cited as leading genetic causes of thrombophilia are shown in Table 5, many of which may be part of a multi-target NGS thrombophilia panel for investigating those with, or at risk of, a venous thrombosis [250–255], as summarised in a 2022 guideline [256].
TABLE 5
| Gene | Product | Location | Pathophysiology |
|---|---|---|---|
| SERPINC1 | Antithrombin | 1q25.1 | Deletion and loss of function SNPs lead to qualitative and quantitative deficiency |
| FV rs6025 | Factor V Leiden G1691A | 1q24.2 | Missense SNP generates a FV resistant to Protein C |
| F2 rs1799963 | Prothrombin G20210A | 11p11.2 | SNP in the 3′ untranslated region leads to increased transcription |
| PROC | Protein C | 2q14.3 | Deficiency leads to failure to inactivate FVa and FVIIIa |
| PROS1 | Protein S | 3q11.1 | Obligate co-factor required for Protein C function |
Genetics of leading forms of thrombophilia.
Platelet Abnormalities
Nurden and Nurden [
257] summarised the genetics of the ontogeny of thrombopoiesis in four stages:
THPO at 3q27.1, coding for thrombopoietin, and MPL at 1p34.2 coding the thrombopoietin receptor (loss of function mutations in either causing congenital amegakaryoctic thrombocytopenia), at the level of the haemopoietic stem cell.
Subsequent roles for HOXA11 at 7p15.2, FLI1 at 11q24.3, RUNX1 at 21q22.2, GATA1 at Xp11.23, GFI1B at 9q34.13, and ETV6 at 12p13.2, all genes coding for a transcription factor, as the megakaryoblast develops.
As the megakaryocyte develops, MYH9 at 22q12.3, coding myosin heavy chain 9, DIAPH1 at 5q31.3, coding diaphanous-related formin, a molecule involved in actin polymerisation, FLNA at Xq28, coding filamin, also with a role in actin structure, and TUBB1 at 20q13.32, coding tubulin, contributing to cytoskeletal integrity, become relevant.
The final stage, that of proplatelet development, involves VWF, ITGA2B (at 17q21.31, coding the integrin CD41/gpIIb), ITGB3 (at 17q21.32, coding CD61/gpIIIa, also an integrin), GP1BA (at 17p13.2, coding CD42b/gpIb-α), GP1BB (at 22q11.21, coding CD42c/gpIb-β), and GP9 (at 3q21.3, coding CD42a/gpIX).
The leading forms of genetic disease of platelets involve surface receptors. In Bernard Soulier syndrome (with a prevalence of 1 per million), there is a thrombocytopenia and a qualitative change, the latter caused by failure of any of GP1BA (the most frequently mutated gene), GP1BB, GP9, or GP5 (at 3q29) to correctly code molecules forming the GpIb-IX-V complex, the receptor for vWf. Failure of this receptor/ligand reaction results in platelets unable to form a thrombus, and so haemorrhage [258, 259]. Glanzmann thrombasthenia also has an incidence of 1/million, but in areas of high consanguinity this may increase five-fold. It is caused by mutations in ITGA2B or ITGB3, that together code for integrins GPIIb/IIIa, also known as the fibrinogen receptor [260]. The molecular pathology of the platelet extends other forms of haemorrhagic disease (Table 6) [257, 261–265]. Gebetsberger and colleagues [266] summarised Sanger, NGS panels, WES and WGS approaches to platelet disorders.
TABLE 6
| Gene | Product | Location | Pathophysiology |
|---|---|---|---|
| RBM8A | An RNA binding protein | 1q21.1 | Thrombocytopenia with absent radius (the forearm bone) |
| SBDS | A ribosome maturation factor | 7q11 | A thrombocytopenia in Schwachman-Diamond syndrome |
| MYH9 | Myosin heavy chain | 22q12.3 | Thrombocytopenia and large platelets, and present as part of the May-Hegglin anomaly, and the syndromes of Sebastian, Epstein and Fechtner |
| WAS | Multiple roles, including actin polymerisation | Xp11.23 | Wiskott-Aldrich syndrome, with thrombocytopenia, small platelets, immunodeficiency And eczema |
| NBEAL2 | Scaffolding protein in alpha granules | 3p21.31 | Grey platelet syndrome: thrombocytopenia |
Genetics of leading platelet disorders.
The Red Blood Cell Membrane and Cytoskeleton
As with the haemoglobinopathies, the geographical distribution of some abnormalities of the erythrocyte membrane and cytoskeleton support the hypothesis that they have developed in response to malaria. Autosomal dominant hereditary elliptocytosis (with an incidence of 0.6%–3% in West Africa) results from mutations in SPTA1 at 1q23.1 or SPTB at 14q23.3, coding for α- or β-spectrin (together, 20% of cases), SLC4A1 at 17q21.31, coding for band 3, the chloride/bicarbonate anion exchanger (15%–20%), or in EPB41 at 1p35.3, coding for another cytoskeletal component, band 4.1 (<15% of cases) [267]. Autosomal recessive hereditary pyropoikilocytosis (also frequently diagnosed in infants of African ancestry) may also be linked to defects in SPTA1 or SPTB, whilst autosomal dominant Southeast Asian ovalocytosis (principally found in Malaysia, Thailand, Indonesia, the Philippines and Papua New Guinea) is caused by a 27-nucleotide deletion in SLC4A1, which confers a resistance to cerebral malaria, with a mean allelic population frequency of ≤1% [268].
Hereditary spherocytosis is the most common hereditary haemolytic anaemia worldwide, and in Caucasians has a prevalence of 0.02%–0.04%. with some 75% being autosomal dominant, although the frequency in China is estimated at 0.014%. The most common causative lesions are in ANK1 at 8p11.21 (e.g., c.-108T>C, c.-153G>A, and c.-204C>G) coding for ankyrin (50%–60% of cases), followed by SLC4A1, SPTA1, SPTB, and EPB42 at 15q15.2, coding for protein 4.2 [269, 270].
Hereditary stomatocytosis is an autosomal-dominant condition where the transport of cations (notably sodium and potassium) is impaired, leading to osmotic misbalance, resulting in two abnormal forms. An overhydrated form is linked to missense variants of RHAG at 6p12.2, coding for Rh-associated glycoprotein (CD241), which functions as an ammonia transporter, whilst over 50% of a dehydrated variant is due to one of three mutations in PIEZO1, at 16q24.2, coding a mechanosensitive cation channel [271, 272]. Another, the Gardos channelopathy, is associated with KCNN4 at 19q13.31, coding a calcium-activated potassium channel. A mild form of stomatocytosis, cryohydrocytosis (present at temperatures <20 °C), results from a missense mutation in SLC4A1 [270, 272].
Iron Overload
The leading disease in this group is hereditary haemochromatosis, the most common such genetic condition in those of European descent, present in its homozygous form in 0.2%–0.33%, with some 9% of the population being heterozygotes. There are several variants. In Type 1, causing 75%–80% of cases, the lesion is in HFE (of 7 exons, and 12 kB long, located within the major histocompatibility complex) at 6p21.3-22.2, with 96% of these being at position 845, generating p.C282Y/p.Cyst282Tyr, and 4% being the p.C282Y/p.Hist63Asp (p.H63D) compound heterozygote genotype. HFE codes for a mature 363 amino product that binds to the transferrin receptor 1 and is also a co-factor for hepcidin synthesis, where loss of function leads to reduced hepcidin mRNA, decreased plasma hepcidin, and so excessive tissue iron accumulation [273, 274].
Type 2a disease is caused by variants of HJV (or HFE2) at 1q21, coding for a molecule linked to the membrane receptor for bone morphogenetic protein (BMP), whilst type 2b concerns HAMP at 19q31, coding for the primary iron regulator molecule hepcidin. BMP6 itself, coded for by BMP6 at 6p24, is a further component of the regulation of hepcidin. Type 3 disease follows from a mutation in TFR2 at 7q22.1, and of 20 kB length, the most common being C>G in exon 6, creating a nonsense mutation of p.Y250X. A case report described a second mutation in exon 2, producing nonsense mutation of p.E60X, both mutations resulting in loss of transferrin receptor 2 [275].
The lesion in type 4 disease (also described as ferroportin disease) is within SCL11A2 at 12q13.12, which codes for ferroportin (FPN), a cell membrane component that regulates the passage of iron from the enterocyte to the plasma, and which itself is regulated by hepcidin. Mutations such as an exon 3 SNP resulting in alanine to aspartic acid at residue 77 (p.A77D), result in an FPN unable to export iron, which therefore accumulates inside the cell. A second from of ferroportin disease is characterised by a SCL11A2 mutation, resulting in a variant of ferroportin that is resistant to regulation by hepcidin and so exports excessive amounts of iron [276–278]. A genome-wide meta-analysis confirmed the importance of HFE, HAMP and SLC11A2 but also reported a possible role for TMPRSS6 at 22q12.3, coding for transmembrane serine protease 6 [279]. Other data from this study pointed to significant odds ratios (95% confidence) for the rs748587164 SNP A/T minor/major allele in TF at 3q22.1, coding for transferrin, of 3.89 (2.46–6.15, p = 5.8 × 10−9), the rs1800562 SNP A/G in HFE of 3.54 (3.29–3.8, p = 2.2 × 10−255), rs1799945 SNP G/! in HFE of 1.44 (1.34–1.55, p = 1.3 × 10−24), and the rs855791 SNP A/G in TMPRSS6 of 0.75 (0.71–0.79, p = 1.9 × 10−26).
Although involved in copper metabolism, autosomal recessive variants in CP at 3q24-25.1, coding for caeruloplasmin, and leading to acaeruloplasminaemia, may also lead to iron overload, almost certainly linked to its ferroxidase activity [280].
Conclusion
Molecular pathology has without doubt provided a revolution is our basic understanding of aetiology, diagnosis and management of numerous diseases, including those of the red blood cell, the haemostasis system, in iron metabolism, and also in blood cancer [281]. One may speculate that genetic testing will continue to be an important and expanding feature of clinical and biomedical science, and conceivably may develop into near-patient testing in primary care and other settings, perhaps even into self-testing. In doing so, disease may be recognised earlier and so addressed with more focus, ideally reducing the need for more complex healthcare referrals.
Statements
Author contributions
All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.
Funding
The author(s) declared that financial support was not received for this work and/or its publication.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.
References
1.
DahmR. Friedrich Miescher and the Discovery of DNA. Dev Biol (2005) 278:274–88. 10.1016/j.ydbio.2004.11.028
2.
BaikieAGJacobsPAMcBrideJAToughIM. Cytogenetic Studies in Acute Leukaemia. Br Med J (1961) 1:1564–71. 10.1136/bmj.1.5239.1564
3.
NowellPCHungerfordDA. Chromosome Studies in Human Leukemia. II. Chronic Granulocytic Leukemia. J Natl Cancer Inst (1961) 27:1013–35.
4.
BlannAD. Recombinant DNA Technology in Haematology. Med Lab Sci (1988) 45:349–57.
5.
NienhuisAWAnagnouNPLeyTJ. Advances in Thalassemia Research. Blood (1984) 63:738–58.
6.
TaherMAminondinSAMasirNAJasmadiNANizamNINShahrulISet alSickle Cell Disease: Understanding Pathophysiology, Clinical Features and Advances in Gene Therapy Approaches. Front Pharmacol (2025) 16:1630994. 10.3389/fphar.2025.1630994
7.
GBD 2017 Causes of Death Collaborators. Global, Regional, and National Age-Sex-Specific Mortality for 282 Cause of Death in 195 Countries and Territories, 1980-2017: A Systematic Analysis for the Global Buden of Disease Study 2017. Lancet (2018) 392:1736–88. 10.1016/S0140-6736(18)32203-7
8.
GBD 2017 Disease and Injury Incidence and Prevalence Collaborators. Global, Regional, and National Incidence, Prevalence, and Years Lived with Disability for 354 Diseases and Injuries for 195 Countries and Territories, 1990–2017: A Systematic Analysis for the Global Burden of Disease Study 2017. Lancet (2018):392;1789–858. 10.1016/S0140-6736(18)32279-7
9.
KohneE. Hemoglobinopathies: Clinical Manifestations, Diagnosis, and Treatment. Dtsch Arztebl Int (2011) 108:532–40. 10.3238/arztebl.2011.0532
10.
GoonasekeraHWPaththinigeCSDissanayakeVHW. Population Screening for Hemoglobinopathies. Annu Rev Genom Hum Genet (2018) 19:355–80. 10.1146/annurev-genom-091416-035451
11.
ChatterjeaJB. Haemoglobinopathies, Glucose-6-Phosphate Dehydrogenase Deficiency and Allied Problems in the Indian Subcontinent. Bull World Health Organ (1966) 35:837–56.
12.
WeatherallDJ. Mapping Haemoglobin Genes. Br Med J (1979) 1:352–4. 10.1136/bmj.2.6186.352
13.
WilsonJTWilsonLBdeRielJKVilla-komaroffLEfstratiadisAForgetBGet alInsertion of Synthetic Copies of Human Globin Genes into Bacterial Plasmids. Nucleic Acid Res (1978) 5:563–81. 10.1093/nar/5.2.563
14.
GilbertJMThorntonAGNienhuisAWAndersonWF. Cell-Free Haemoglobin Synthesis in Beta-Thalassaemia. Proc Natl Acad Sci USA (1970) 67:1854–61. 10.1073/pnas.67.4.1854
15.
BenzEJForgetBG. Defect in Messenger RNA for Haemoglobin Synthesis in Beta Thalassaemia. J Clin Invest (1971) 50:2755–60. 10.1172/JCI106778
16.
WeatherallDJ. Molecular Pathology of the Thalassaemia Disorders. West J Med (1976) 124:388–402.
17.
TaylorJMDozyAKanYWVarmusHELie-InjoLEGanesanJet alGenetic Lesion in Homozygous Alpha Thalassaemia (Hydrops fetalis). Nature (1974) 251:392–3. 10.1038/251392a0
18.
OttolenghiSLanyonWGPaulJWilliamsonRWeatherallDJCleggJBet alThe Severe Form of Alpha Thalassaemia Is Caused by a Haemoglobin Gene Deletion. Nature (1974) 251:389–92. 10.1038/251389a0
19.
OttolenghiSLanyonWGWilliamsonRWeatherallDJCleggJBPitcherCS. Human Globin Gene Analysis for a Patient With Betao/Delta Beta-Thalassemia. Proc Natl Acad Sci U S A. (1975) 72:2294–9. 10.1073/pnas.72.6.2294
20.
OldJMProudfootNJWoodWGLongleyJICleggJBWeatherallDJ. Characterization of Beta-Globin mRNA in the Beta0-Thalassaemias. Cell (1978) 14:289–98. 10.1016/0092-8674(78)90115-0
21.
HiggsDRPressleyLOldJMHuntDMCleggJBWeatherallDJet alNegro Alpha-Thalassaemia Is Caused by Deletion of a Single Alpha-Globin Gene. Lancet (1979) 2:272–6. 10.1016/s0140-6736(79)90290-3
22.
OrkinSHOldJMWeatherallDJNathanDG. Partial Deletion of Beta-Globin DNA in Certain Patients with Betao-Thalassaemia. Proc Natl Acad Sci USA (1979) 76:2400–4. 10.1073/pnas.76.5.2400
23.
LauerJShenCKManiatisT. The Chromosomal Arrangement of Human Alpha-Like Globin Genes: Sequence Homology and Alpha-Globin Gene Deletions. Cell (1980) 20:119–30. 10.1016/0092-8674(80)90240-8
24.
PressleyLHiggsDRCleggJBWeatherallDJ. Gene Deletions in α-Thalassaemia Prove that the 5’ ζ Locus Is Functional. Proc Natl Acad Sci USA (1980) 77:3586–9. 10.1073/pnas.77.6.3586
25.
PressleyLHiggsDRAldridgeBMetaxatou-MavromatiACleggJBWeatherallDJ. Characterisation of a New α-Thalassaemia 1 Defect due to a Partial Deletion of the α Globin Gene Complex. Nucleic Acid Res (1980) 8:4889–98. 10.1093/nar/8.21.4889
26.
KanYWDozyAMVarmusHETaylorJMHollandJPLie-InjoLEet alDeletion of Alpha-Globin Genes in Haemoglobin-H Disease Demonstrates Multiple Alpha-Globin Structural Loci. Nature (1975) 255:255–6. 10.1038/255255a0
27.
KattamisCMetaxotou-MavromatiATsiatraEMetaxatouCWasiPWoodWGet alHaemoglobin Bart’s Hydrops Syndrome in Greece. Br Med J (1980) 281:268–70. 10.1136/bmj.281.6235.268
28.
HiggsDRPressleyLAldridgeBCleggJBWeatherallDJCaoAet alGenetic and Molecular Diversity in Nondeletion HbH Disease. Proc Natl Acid Sci USA (1981) 78:5833–7. 10.1073/pnas.78.9.5833
29.
BenzEJGlassJTsistrakisGAHillmanDGCavallescoCCoupalEet alHeterogeneity of Messenger RNA Defects in the Thalassaemia Syndromes. Annals NY Acad Sci (1980) 344:101–12. 10.1111/j.1749-6632.1980.tb33653.x
30.
BaralleFEShouldersCCGoodbournSJeffreysAProudfootNJ. The 5’flanking Region of the Human Epsilon-Globin Gene. Nucleic Acids Res (1980) 8:4393–404. 10.1093/nar/8.19.4393
31.
TakiharaYNakamuraTYamadaHTakagiYFukumakiY. A Novel Mutation in the TATA Box in a Japanese Patient with Beta+-Thalassaemia. Blood (1986) 67(2):547–50.
32.
GeraldPSDiamondLK. A New Hereditary Hemoglobinopathy (The Lepore Trait) and Its Interaction with Thalassemia Trait. Blood (1958) 13:835–44.
33.
FlavellRAKooterJMDe BoerELittlePFWilliamsonR. Analysis of the Beta-Delta-Globin Gene Loci in Normal and Hb Lepore DNA: Direct Determination of Gene Linkage and Intergene Distance. Cell (1978) 15:25–41. 10.1016/0092-8674(78)90080-6
34.
OrkinSH. The Duplicated Human α-Globin Genes Lie Close Together in Cellular DNA. Proc Natl Acid Sci USA (1978) 75:5950–4. 10.1073/pnas.75.12.5950
35.
JacksonIJWilliamsonR. Mapping of the Human Globin Genes. Br J Haematol (1980) 46:341–9. 10.1111/j.1365-2141.1980.tb05980
36.
DeisserothANienhuisATurnerPVelezRAndersonWFRuddleFet alLocalization of the Human Alpha-Globin Structural Gene to Chromosome 16 in Somatic Cell Hybrids by Molecular Hybridization Assay. Cell (1977) 12:205–18. 10.1016/0092-8674(77)90198-2
37.
DeisserothANienhuisALawrenceJGilesRTurnerPRuddleFH. Chromosomal Localization of Human Beta Globin Gene on Human Chromosome 11 in Somatic Cell Hybrids. Proc Natl Acad Sci USA (1978) 75:1456–60. 10.1073/pnas.75.3.1456
38.
BuckleVJHiggsDRWilkieAOSuperMWeatherallDJ. Localisation of Human Alpha Globin to 16p13.3----pter. J Med Genet (1988) 25:847–9. 10.1136/jmg.25.12.847
39.
LinCCDraperPNDe BraekeleerM. High Resolution Chromosomal Location of the β-Gene of the Human β-Globin Gene Complex by in situ Hybridisation. Cytogenet Cell Genet (1985) 39:269–74. 10.1159/000132156
40.
FlavellRABernardsRKooterJMde BoerELittlePFAnnisonGet alThe Structure of the Human Beta-Globin Gene in Beta-Thalassaemia. Nucleic Acids Res (1979) 6:2749–60. 10.1093/nar/6.8.2749
41.
LittlePFWhitelawEAnnisonGWilliamsonRKooterJMFlavellRAet alThe Detection and Use of Hemoglobin Mutants in the Direct Analysis of Human Globin Genes. Blood (1980) 55:1060–2.
42.
BernardsRLittlePFAnnisonGWilliamsonRFlavellRA. Structure of the Human G Gamma-A Gamma-Delta-Beta-Globin Gene Locus. Proc Natl Acad Sci USA (1979) 76:4827–31. 10.1073/pnas.76.10.4827
43.
AtwehGFWongCReedRAntonarakisSEZhuDGhoshPKet alA New Mutation in ICS-1 of the Human β-Globin Gene Causing β-Thalassaemia due to Abnormal Splicing. Blood (1987) 70:147–51.
44.
MetherallJECollinsFSPanJWeissmanSMForgetBG. Beta Zero Thalassemia Caused by a Base Substitution that Creates an Alternative Splice Acceptor Site in an Intron. EMBO J (1986) 5:2551–7. 10.1002/j.1460-2075.1986.tb04534.x
45.
AtwehGFAnagnouNPShearinJForgetBGKaufmanRE. Beta-Thalassemia Resulting from a Single Nucleotide Substitution in an Acceptor Splice Site. Nucleic Acids Res (1985) 13:777–90. 10.1093/nar/13.3.777
46.
LiebhaberSAColemanMBAdamsJGCashFESteinbergMH. Molecular Basis for Nondeletion Alpha-Thalassemia in American Blacks. Alpha 2(116GAG----UAG). J Clin Invest (1987) 80:154–9. 10.1172/JCI113041
47.
ProudfootNJBaralleFE. Molecular Cloning of Human Epsilon-Globin Gene. Proc Natl Acad Sci USA (1979) 76:5435–9. 10.1073/pnas.76.11.5435
48.
LigtenbergMJGennissenAMVosHLHilkensJ. A Single Nucleotide Polymorphism in an Exon Dictates Allele Dependent Differential Splicing of Episialin mRNA. Nucleic Acids Res (1991) 19:297–301. 10.1093/nar/19.2.297
49.
OldJMWardRHPetrouMKaragözluFModellBWeatherallDJ. First-Trimester Fetal Diagnosis for Haemoglobinopathies: Three Cases. Lancet (1982) 2:1413–6. 10.1016/s0140-6736(82)91324-1
50.
OrkinSHMarkhamAFKazazianHH. Direct Detection of the Common Mediterranean β-Thalassaemia Gene with Synthetic DNA Probes. J Clin Invest (1983) 71:775–9. 10.1172/jci110826
51.
BoehmCDAntonarakisSEPhillipsJAStettenGKazazianHH. Prenatal Diagnosis Using DNA Polymorphisms. Report on 95 Pregnancies at Risk for Sickle-Cell Disease or Beta-Thalassemia. N Engl J Med (1983) 308:1054–8. 10.1056/NEJM198305053081803
52.
OrkinSH. Prenatal Diagnosis of Haemoglobin Disorders by DNA Analysis. Blood (1984) 63:249–53.
53.
PirastuMKanYWCaoAConnerBJTeplitzRLWallaceRB. Prenatal Diagnosis of Beta-Thalassemia. Detection of a Single Nucleotide Mutation in DNA. N Engl J Med (1983) 309(5):284–7. 10.1056/NEJM198308043090506
54.
AlterBP. Advances in the Prenatal Diagnosis of Haematological Diseases. Blood (1984) 64:329–40.
55.
CongoteLFHamiltonEFChowJCPerryTB. Prenatal Diagnosis of Hemoglobinopathies: Evaluation of Techniques for Analysing Globin-Chain Synthesis in Blood Samples Obtained by Fetoscopy. Can Med Assoc J. (1982) 127:843–9.
56.
AlterBPStumpDD. Prenatal Diagnosis of Hemoglobinopathies: A Potential Application of High-Performance Liquid Chromatography. Hemoglobin (1987) 11:341–52. 10.3109/03630268709042853
57.
Rouyer-FessardPBeuzardYVidaudMJohnPMibashanRS. Prenatal Diagnosis of Beta Thalassaemia by Reverse Phase HPLC. Prenat Diagn (1987) 7:171–8. 10.1002/pd.1970070304
58.
HiggsDRVickersMAWilkieAOMPretoriusIMJarmanAPWeatherallDJ. A Review of the Molecular Genetics of the Human α-Globin Gene Cluster. Blood (1989) 73:1081–104.
59.
CaoAMurruS. Molecular Pathology and Detection of Beta-Thalassemias. Prog Clin Biol Res. (1989) 309:3–11.
60.
ConstantoulakisPJosephsonBMangahasLPapayannopoulouTEnverTCostantiniFet alLocus Control region-A Gamma Transgenic Mice: A New Model for Studying the Induction of Fetal Hemoglobin in the Adult. Blood (1991) 77(6):1326–33.
61.
OrkinSH. Regulation of Globin Gene Expression in Erythroid Cells. Eur J Biochem (1995) 231:271–81. 10.1111/j.1432-1033.1995.tb20697.x
62.
LeeJSLeeCHChungJH. The β-Globin Promoter Is Important for Recruitment of Erythroid Krüppel-Like Factor to the Locus Control Region in Erythroid Cells. Proc Natl Acad Sci U S A (1999) 96(18):10051–5. 10.1073/pnas.96.18.10051
63.
LangdonSDKaufmanRE. Gamma-Globin Gene Promoter Elements Required for Interaction with Globin Enhancers. Blood (1998) 91(1):309–18.
64.
Available online at: https://www.ncbi.nlm.nih.gov/gene/109580095. (Accessed 11th February 2026).
65.
SchoutenJPMcElgunnCJRaymond WaaijerRZwijnenburgDDiepvensFPalsG. Relative Quantification of 40 Nucleic Acid Sequences by Multiplex Ligation-Dependent Probe Amplification. Nucleic Acids Res (2002) 30(12):e57. 10.1093/nar/gnf056
66.
MirasenaSShimbhuDSanguansermsriMSanguansermsriT. Detection of Beta-Thalassemia Mutations Using a Multiplex Amplification Refractory Mutation System Assay. Hemoglobin (2008) 32(4):403–9. 10.1080/03630260701798391
67.
HarteveldCLRefaldiCCassinerioECappelliniMDGiordanoPC. Segmental Duplications Involving the Alpha-Globin Gene Cluster Are Causing Beta-Thalassemia Intermedia Phenotypes in Beta-Thalassemia Heterozygous Patients. Blood Cells Mol Dis (2008) 40(3):312–6. 10.1016/j.bcmd.2007.11.006
68.
SollainoMCPagliettiMEPerseuLGiaguNLoiDGalanelloR. Association of α Globin Gene Quadruplication and Heterozygous β Thalassemia in Patients with Thalassemia Intermedia. Haematologica (2009) 94(10):1445–8. 10.3324/haematol.2009.005728
69.
PapasavvaTvan IjckenWFKockxCEvan den HoutMCGNKountourisPKythreotisLet alNext Generation Sequencing of Snps for Non-Invasive Prenatal Diagnosis: Challenges and Feasibility as Illustrated by an Application to β-Thalassaemia. Eur J Hum Genet (2013) 21(12):1403–10. 10.1038/ejhg.2013.47
70.
HeJSongWYangJLuSYuanYGuoJet alNext-Generation Sequencing Improves Thalassemia Carrier Screening Among Premarital Adults in a High Prevalence Population: The Dai Nationality, China. Genet Med (2017) 19(9):1022–31. 10.1038/gim.2016.218
71.
LoYMTeinMSLauTKHainesCJLeungTNPoonPMet alQuantitative Analysis of Fetal DNA in Maternal Plasma and Serum: Implications for Noninvasive Prenatal Diagnosis. Am J Hum Genet (1998) 62:768–75. 10.1086/301800
72.
MonniGCauGUsaiVPerraGLaiRIbbaGet alPreimplantation Genetic Diagnosis for Beta-Thalassaemia: The Sardinian Experience. Prenat. Diagn. (2004) 24:949–54. 10.1002/pd.1051
73.
SaikiRKBugawanTLHornGTMullisKBErlichHA. Analysis of Enzymatically Amplified Beta-Globin and HLA-DQ Alpha DNA with Allele-Specific Oligonucleotide Probes. Nature (1986) 324:163–6. 10.1038/324163a0
74.
WongFCKDennisYMLoD. Prenatal Diagnosis Innovation: Genome Sequencing of Maternal Plasma. Annu Rev Med (2016) 67:419–32. 10.1146/annurev-med-091014-115715
75.
MonniGPeddesCIuculanoAIbbaRM. From Prenatal to Preimplantation Genetic Diagnosis of β-Thalassemia. Prevention Model in 8748 Cases: 40 Years of Single Center Experience. J Clin Med (2018) 7:35. 10.3390/jcm7020035
76.
GrimholtRMUrdalPKlingenbergOPiehlerAP. Rapid and Reliable Detection of α-Globin Copy Number Variations by Quantitative Real-Time PCR. BMC Hematol. (2014) 14:4. 10.1186/2052-1839-14-4
77.
BaoXQinDMaJZhouXWangJYaoCet alAccurate Detection of α-Globin Gene Copy Number Variants with Two Reactions Using Droplet Digital PCR. Hematology (2022) 27:198–203. 10.1080/16078454.2022.2030885
78.
SankaranVGGallagherPG. Applications of High-Throughput DNA Sequencing to Benign Hematology. Blood (2013) 122:3575–82. 10.1182/blood-2013-07-460337
79.
Steinberg-ShemerOUlirschJCNoy-LotanSKrasnovTAttiasDDganyOet alWhole-Exome Sequencing Identifies an α-Globin Cluster Triplication Resulting in Increased Clinical Severity of β-Thalassemia. Cold Spring Harb Mol Case Stud (2017) 3:a001941. 10.1101/mcs.a001941
80.
SchadtEETurnerSKasarskisA. A Window into Third-Generation Sequencing. Hum Mol Genet (2010) 19:R227–40. 10.1093/hmg/ddq416
81.
OhshiroTMatsubaraKTsutsuiMFuruhashiMTaniguchiMKawaiT. Single-Molecule Electrical Random Resequencing of DNA and RNA. Sci Rep (2012) 2:501. 10.1038/srep00501
82.
LeBlancVGMarraMA. Next-Generation Sequencing Approaches in Cancer: Where Have They Brought Us and Where Will They Take Us?Cancers (2015) 7:1925–58. 10.3390/cancers7030869
83.
PengCZhangHRenJChenHDuZZhaoTet alAnalysis of Rare Thalassemia Genetic Variants Based on Third-Generation Sequencing. Sci Rep (2022) 12:9907. 10.1038/s41598-022-14038-8
84.
LongJSunLGongFZhangCMaoALuYet alThird-Generation Sequencing: A Novel Tool Detects Complex Variants in the α-Thalassemia Gene. Gene (2022) 822:146332. 10.1016/j.gene.2022.146332
85.
LiuSWangHLeighDCramDSWangLYaoY. Third-Generation Sequencing: Any Future Opportunities for PGT?J Assist Reprod Genet. (2021) 38:357–64. 10.1007/s10815-020-02009-9
86.
HuangWQuSQinQYangXHanWLaiYet alNanopore Third-Generation Sequencing for Comprehensive Analysis of Hemoglobinopathy Variants. Clin Chem (2023) 69:1062–71. 10.1093/clinchem/hvad073
87.
HassanSBaharRJohanMFMohamed HashimEKAbdullahWZEsaEet alNext-Generation Sequencing (NGS) and Third-Generation Sequencing (TGS) for the Diagnosis of Thalassaemia. Diagnosis (2023) 13:373. 10.3390/diagnostics13030373
88.
StamatoyannopoulosJA. Future Prospects for Treatment of Hemoglobinopathies. West J Med (1992) 157:631–6.
89.
QuekLTheinSL. Molecular Therapies in Beta-Thalassaemia. Br J Haematol (2007) 136:353–65. 10.1111/j.1365-2141.2006.06408.x
90.
JansenREmbdenJDGaastraWSchoulsLM. Identification of Genes that Are Associated with DNA Repeats in Prokaryotes. Mol Microbiol (2002) 43:1565–75. 10.1046/j.1365-2958.2002.02839.x
91.
JinekMChylinskiKFonfaraIHauerMDoudnaJACharpentierE. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science (2012) 337:816–21. 10.1126/science.1225829
92.
DoudnaJACharpentierE. Genome Editing. the New Frontier of Genome Engineering with CRISPR-Cas9. Science (2014) 346:1258096. 10.1126/science.1258096
93.
XieFYeLChangJCBeyerAIWangJMuenchMOet alSeamless Gene Correction of β-Thalassemia Mutations in Patient-Specific iPSCs Using CRISPR/Cas9 and piggyBac. Genome Res. (2014) 24:1526–33. 10.1101/gr.173427.114
94.
CaiLBaiHMahairakiVGaoYHeCWenYet alA Universal Approach to Correct Various HBB Gene Mutations in Human Stem Cells for Gene Therapy of Beta-Thalassemia and Sickle Cell Disease. Stem Cells Transl Med (2018) 7:87–97. 10.1002/sctm.17-0066
95.
SatterwhiteESonokiTWillisTGHarderLNowakRArriolaELet alThe BCL11 Gene Family: Involvement of BCL11A in Lymphoid Malignancies. Blood (2001) 98:3413–20. 10.1182/blood.v98.12.3413
96.
UdaMGalanelloRSannaSLettreGSankaranVGChenWet alGenome-Wide Association Study Shows BCL11A Associated with Persistent Fetal Hemoglobin and Amelioration of the Phenotype of Beta-Thalassemia. Proc Natl Acad Sci U S A (2008) 105:1620–5. 10.1073/pnas.0711566105
97.
SedgewickAETimofeevNSebastianiPSoJCCMaESKChanLCet alBCL11A Is a Major HbF Quantitative Trait Locus in Three Different Populations with Beta-Hemoglobinopathies. Blood Cells Mol Dis (2008) 41:255–8. 10.1016/j.bcmd.2008.06.007
98.
SankaranVGMenneTFXuJAkieTELettreGVan HandelBet alHuman Fetal Hemoglobin Expression Is Regulated by the Developmental Stage-Specific Repressor BCL11A. Science (2008) 322:1839–42. 10.1126/science.1165409
99.
FrangoulHAltshulerDCappelliniMDChenYSDommJEustaceBKet alCRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia. N Engl J Med (2021) 384:252–60. 10.1056/NEJMoa2031054
100.
FuBLiaoJChenSLiWWangQHuJet alCRISPR-Cas9-Mediated Gene Editing of the BCL11A Enhancer for Pediatric β0/β0 Transfusion-Dependent β-Thalassemia. Nat Med (2022) 28:1573–80. 10.1038/s41591-022-01906-z
101.
QuaglianoAAcevedoDHardiganPPrasadS. Using Clustered Regularly Interspaced Shirt Palindromic Repeats Gene Editing to Induce Permanent Expression of Foetal Haemoglobin in β-Thalassaemia and Sickle Cell Disease. A Comparative meta-analysis. Front Med (2022) 9:943631. 10.3389/fmed.2022.943631
102.
HawksworthJSatchwellTJMeindersMDanielsDEReganFThorntonNMet alEnhancement of Red Blood Cell Transfusion Compatibility Using CRISPR-Mediated Erythroblast Gene Editing. EMBO Mol Med (2018) 10:e8454. 10.15252/emmm.201708454
103.
ModellBDarlisonM. Global Epidemiology of Haemoglobin Disorders and Derived Service Indicators. Bull World Health Organ (2008) 86:480–7. 10.2471/blt.06.036673
104.
WilliamsTNWeatherallDJ. World Distribution, Population Genetics, and Health Burden of the Hemoglobinopathies. Cold Spring Harb Perspect Med. (2012) 2:a011692. 10.1101/cshperspect.a011692
105.
GBD 2021 Sickle Cell Disease Collaborators. Global, Regional, and National Prevalence and Mortality Burden of Sickle Cell Disease, 2000–2021: A Systematic Analysis from the Global Burden of Disease Study 2021. Lancet Haematol (2023) 10:e585–99. 10.1016/S2352-3026(23)00118-7
106.
AllisonAC. Protection Afforded by Sickle-Cell Trait Against Subtertian Malareal Infection. Br Med J. (1954) 1:290–4. 10.1136/bmj.1.4857.290
107.
Anonymous. Sickling and Malaria. Br Med J. (1954) 1:320–1.
108.
SerjeantGR. One Hundred Years of Sickle Cell Disease. Br J Haematol (2010) 151:425–9. 10.1111/j.1365-2141.2010.08419.x
109.
Hall-CraggsMMarsdenPDRaperABLehmannHBealeD. Homozygous Sickle-Cll Anaemia Arising from Two Different Haemoglobins. Br Med J (1964) 2:87–9. 10.1136/bmj.2.5401.84
110.
MurayamaM. Molecular Mechanism of Red Cell Sickling. Science (1966) 153:145–9. 10.1126/science.153.3732.145
111.
BirdGW. The Haemoglobinopathies. Br Med J (1972) 1:363–8. 10.1136/bmj.1.5796.363
112.
KendallAGOjwangPJSchroederWAHuismanTH. Hemoglobin Kenya, the Product of a Gamma-Beta Fusion Gene: Studies of the Family. Am J Hum Genet (1973) 25:548–63.
113.
KanYWDozyAM. Polymorphism of DNA Sequence Adjacent to Human Beta-Globin Structural Gene: Relationship to Sickle Mutation. Proc Natl Acad Sci U S A (1978) 75:5631–5. 10.1073/pnas.75.11.5631
114.
OrkinSH. Selective Restriction Endonuclease Cleavage of Human Globin Genes. J Biol Chem. (1978) 253:12–5.
115.
FeldenzerJMearsJGBurnsALNattaCBankA. Heterogeneity of DNA Fragments Associated with the Sickle-Globin Gene. J Clin Invest (1979) 64:751–5. 10.1172/JCI109519
116.
MarottaCAWilsonJTForgetBGWeissmanSM. Human Beta-Globin Messenger RNA. III. Nucleotide Sequences Derived from Complementary DNA. J Biol Chem (1977) 252:5040–53.
117.
GeeverRFWilsonLBNallasethFSMilnerPFBittnerMWilsonJT. Direct Identification of Sickle Cell Anemia by Blot Hybridization. Proc Natl Acad Sci U S A. (1981) 78:5081–5. 10.1073/pnas.78.8.5081
118.
KanYWDozyAM. Antenatal Diagnosis of Sickle-Cell Anaemia by D.N.A. Analysis of Amniotic-Fluid Cells. Lancet (1978) 2:910–2. 10.1016/s0140-6736(78)91629-x
119.
PhillipsJAPannySRKazazianHHJrBoehmCDScottAFSmithKD. Prenatal Diagnosis of Sickle Cell Anemia by Restriction and Endonuclease Analysis: Hindiii Polymorphisms in Gamma-Globin Genes Extend Test Applicability. Proc Natl Acad Sci U S A (1980) 77:2853–6. 10.1073/pnas.77.5.2853
120.
BenzEJJr. Molecular Genetics of the Sickling Syndromes: Evolution of New Strategies for Improved Diagnosis. Am J Pediatr Hematol Oncol (1984) 6:59–66.
121.
GilmanJGHuismanTH. Two Independent Genetic Factors in the Beta-Globin Gene Cluster Are Associated with High G Gamma-Levels in the Hbf of SS Patients. Blood (1984) 64:452–7.
122.
SheldonEKelloggDELevensonCBlochWAldwinLBirchDet alNonisotopic M13 Probes for Detecting the Beta-Globin Gene: Application to Diagnosis of Sickle Cell Anemia. Clin Chem (1987) 33:1368–71.
123.
GarbuttGJWilsonJTSchusterGSLearyJJWardDC. Use of Biotinylated Probes for Detecting Sickle Cell Anemia. Clin Chem (1985) 31:1203–6.
124.
EmburySHScharfSJSaikiRKGholsonMAGolbusMArnheimNet alRapid Prenatal Diagnosis of Sickle Cell Anaemia by a New Method of DNA Analysis. N Engl J Med (1987) 316:656–61. 10.1056/NEJM198703123161103
125.
KulozikAELyonsJKohneEBartramCRKleihauerE. Rapid and Non-Radioactive Prenatal Diagnosis of Beta Thalassaemia and Sickle Cell Disease: Application of the Polymerase Chain Reaction (PCR). Br J Haematol (1988) 70:455–8. 10.1111/j.1365-2141.1988.tb02516.x
126.
WuDYNozariGSchöldMConnerBJWallaceRB. Direct Analysis of Single Nucleotide Variation in Human DNA and RNA Using in situ Dot Hybridization. DNA (1989) 8:135–42. 10.1089/dna.1.1989.8.135
127.
CrisanDMattsonJC. Amplification of Intermediate-Size DNA Sequences from Formalin and B-5 Fixed Tissue by Polymerase Chain Reaction. Clin Biochem (1992) 25:99–103. 10.1016/0009-9120(92)80051-h
128.
BirikhKRPlutalovOVSchwartzEIDeviPSBerlinYA. A Modified Approach to Identification of the Sickle Cell Anaemia Mutation by Means of Allele-Specific Polymerase Chain Reaction. Hum Mutat (1992) 1:417–9. 10.1002/humu.1380010511
129.
Fischel-GhodsianNHirschPCBohlmanMC. Rapid Detection of the Hemoglobin C Mutation by Allele-Specific Polymerase Chain Reaction. Am J Hum Genet (1990) 47:1023–4.
130.
StamatoyannopoulosJA. Future prospects for treatment of hemoglobinopathies. West J Med (1992) 157:631–6.
131.
WoodNStandenGHowsJBradleyBBidwellJ. Diagnosis of sickle-cell Disease with a Universal Heteroduplex Generator. Lancet (1993) 342:1519–20. 10.1016/s0140-6736(05)80086-8
132.
WoodNStandenGOldJBidwellJ. Optimisation and Properties of a UHG for Genotyping of Hemoglobins S and C. Hum Mutat (1995) 5:166–72.
133.
GurgeyAMesciLOzerkamKOnerC. Analysis of Chimerism by PCR Using Mutant-Specific Primers and Sequencing of B-Gene Region After Allogenic Bone Marrow Transplantation in Sickle Cell Anemia. Am J Hematol (1995) 50:318–9. 10.1002/ajh.2830500425
134.
TangDCEbbDHardisonRCRodgersGP. Restoration of the CCAAT Box or Insertion of the CACCC Motif Activates [Corrected] Delta-Globin Gene Expression. Blood (1997) 90:421–7.
135.
GräslundTLiXMagnenatLPopkovMBarbasCF. Exploring Strategies for the Design of Artificial Transcription Factors: Targeting Sites Proximal to Known Regulatory Regions for the Induction of Gamma-Globin Expression and the Treatment of Sickle Cell Disease. J Biol Chem (2005) 280:3707–14. 10.1074/jbc.M406809200
136.
BankA. Regulation of Human Fetal Hemoglobin: New Players, New Complexities. Blood (2006) 107:435–43. 10.1182/blood-2005-05-2113
137.
HoCWillisBFShenTJDazhenNTSunDPTamMFet alRoles of Alpha 114 and Beta 87 Amino Acid Residues in the Polymerization of Hemoglobin S: Implications for Gene Therapy. J Mol Biol (1996) 263:475–85. 10.1006/jmbi.1996.0590
138.
Cole-StraussAYoonKXiangYByrneBCRiceMCGrynJet alCorrection of the Mutation Responsible for Sickle Cell Anemia by an RNA-DNA Oligonucleotide. Science (1996) 273:1386–9doi. 10.1126/science.273.5280.1386
139.
PawliukRWestermanKAFabryMEPayenETigheRBouhassiraEEet alCorrection of Sickle Cell Disease in Transgenic Mouse Models by Gene Therapy. Science (2001) 294:2368–71. 10.1126/science.1065806
140.
LevasseurDNRyanTMPawlikKMTownesTM. Correction of a Mouse Model of Sickle Cell Disease: Lentiviral/Antisickling Beta-Globin Gene Transduction of Unmobilized, Purified Hematopoietic Stem Cells. Blood (2003) 102:4312–9. 10.1182/blood-2003-04-1251
141.
ImrenSFabryMEWestermanKAPawliukRTangPRostenPMet alHigh-Level Beta-Globin Expression and Preferred Intragenic Integration After Lentiviral Transduction of Human Cord Blood Stem Cells. J Clin Invest (2004) 114:953–62. 10.1172/JCI21838
142.
LiebermanJSongELeeSKShankarP. Interfering with Disease: Opportunities and Roadblocks to Harnessing RNA Interference. Trends Mol Med (2003) 9(9):397–403. 10.1016/s1471-4914(03)00143-6
143.
DykxhoornDMSchlehuberLDLondonIMLiebermanJ. Determinants of Specific RNA interference-mediated Silencing of Human Beta-Globin Alleles Differing by a Single Nucleotide Polymorphism. Proc Natl Acad Sci U S A. (2006) 103:5953–8. 10.1073/pnas.0601309103
144.
VasavdaNUlugPKondaveetiSRamasamyKSugaiTCheungGet alCirculating DNA: A Potential Marker of Sickle Cell Crisis. Br J Haematol (2007) 139:331–6. 10.1111/j.1365-2141.2007.06775.x
145.
UlugPVasavdaNKumarRKeirLAwogbadeMCunninghamJet alHydroxyurea Therapy Lowers Circulating DNA Levels in Sickle Cell Anemia. Am J Hematol (2008) 83:714–6. 10.1002/ajh.21237
146.
SebastianiPWangLNolanVGMelistaEMaQBaldwinCTet alFetal Hemoglobin in Sickle Cell Anemia: Bayesian Modelling of Genetic Associations. Am J Hematol (2008) 83:189–95. 10.1002/ajh.21048
147.
ZhouDLiuKSunCWPawlikKMTownesTM. KLF1 Regulates BCL11A Expression and Gamma-to Beta-Globin Gene Switching. Nat Genet (2010) 42:742–4. 10.1038/ng.637
148.
GhediraESLecerfLFaubertECostesBMoradkhaniKBachirDet alEstimation of the Difference in HbF Expression due to Loss of the 5' δ-Globin BCL11A Binding Region. Haematologica (2013) 98:305–8. 10.3324/haematol.2012.061994
149.
ChenZLuoHYBasranRKHsuTHMangDWHNuntakarnLet alA T-to-G Transversion at Nucleotide -567 Upstream of HBG2 in a GATA-1 Binding Motif Is Associated with Elevated Hemoglobin F. Mol Cell Biol (2008) 28:4386–93. 10.1128/MCB.00071-08
150.
SolovieffNMiltonJNHartleySWShervaRSebastianiPDworkisDAet alFetal Hemoglobin in Sickle Cell Anemia: Genome-Wide Association Studies Suggest a Regulatory Region in the 5' Olfactory Receptor Gene Cluster. Blood (2010) 115:1815–22. 10.1182/blood-2009-08-239517
151.
KumkhaekCTaylorJGZhuJHoppeCKatoGJRodgersGP. Fetal Haemoglobin Response to Hydroxycarbamide Treatment and sar1a Promoter Polymorphisms in Sickle Cell Anaemia. Br J Haematol (2008) 141:254–9. 10.1111/j.1365-2141.2008.07045.x
152.
AkinsheyeIAlsultanASolovieffNNgoDBaldwinCTSebastianiPet alFetal Hemoglobin in Sickle Cell Anemia. Blood (2011) 118:19–27. 10.1182/blood-2011-03-325258
153.
RomeroZUrbinatiFGeigerSCooperARWherleyJKaufmanMLet alβ-Globin Gene Transfer to Human Bone Marrow for Sickle Cell Disease. J Clin Invest (2013) 123(8):3317–30. 10.1172/JCI67930
154.
SjeklochaLMWongPYBelcherJDVercellottiGMSteerCJ. β-Globin Sleeping Beauty Transposon Reduces Red Blood Cell Sickling in a Patient-Derived CD34(+)-Based In Vitro Model. PLoS One (2013) 8(11):e80403. 10.1371/journal.pone.0080403
155.
VoitRAHendelAPruett-MillerSMPorteusMH. Nuclease-Mediated Gene Editing by Homologous Recombination of the Human Globin Locus. Nucleic Acids Res (2014) 42(2):1365–78. 10.1093/nar/gkt947
156.
HobanMDLumaquinDKuoCYRomeroZLongJHoMet alCRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ Cells. Mol Ther (2016) 24(9):1561–9. 10.1038/mt.2016.148
157.
BrendelCGudaSRenellaRBauerDECanverMCKimYJet alLineage-Specific BCL11A Knockdown Circumvents Toxicities and Reverses Sickle Phenotype. J Clin Invest (2016) 126(10):3868–78. 10.1172/JCI87885
158.
YeLWangJTanYBeyerAIXieFMuenchMOet alGenome Editing Using CRISPR-Cas9 to Create the HPFH Genotype in HSPCs: An Approach for Treating Sickle Cell Disease and β-thalassemia. Proc Natl Acad Sci U S A (2016) 113(38):10661–5. 10.1073/pnas.1612075113
159.
AntonianiCMeneghiniVLattanziAFelixTRomanoOMagrinEet alInduction of Fetal Hemoglobin Synthesis by CRISPR/Cas9-Mediated Editing of the Human β-Globin Locus. Blood (2018) 131(17):1960–73. 10.1182/blood-2017-10-811505
160.
DeverDPBakROReinischACamarenaJWashingtonGNicolasCEet alCRISPR/Cas9 β-Globin Gene Targeting in Human Haematopoietic Stem Cells. Nature (2016) 539(7629):384–9. 10.1038/nature20134
161.
Lamsfus-CalleADaniel-MorenoAAntonyJSEptingTHeumosLBaskaranPet alComparative Targeting Analysis of KLF1, BCL11A, and HBG1/2 in CD34+ HSPCs by CRISPR/Cas9 for the Induction of Fetal Hemoglobin. Sci Rep (2020) 10(1):10133. 10.1038/s41598-020-66309-x
162.
RibeilJAHacein-Bey-AbinaSPayenEMagnaniASemeraroMMagrinEet alGene Therapy in a Patient with Sickle Cell Disease. N Engl J Med (2017) 376(9):848–55. 10.1056/NEJMoa1609677
163.
EsrickEBLehmannLEBiffiAAchebeMBrendelCCiuculescuMFet alPost-Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease. N Engl J Med (2021) 384:205–15. 10.1056/NEJMoa2029392
164.
KanterJWaltersMCKrishnamurtiLMaparaMYKwiatkowskiJLRifkin-ZenenbergSet alBiologic and Clinical Efficacy of LentiGlobin for Sickle Cell Disease. N Engl J Med (2022) 386(7):617–28. 10.1056/NEJMoa2117175
165.
MagrinESemeraroMHebertNJosephLMagnaniAChalumeauAet alLong-Term Outcomes of Lentiviral Gene Therapy for the β-hemoglobinopathies: The HGB-205 Trial. Nat Med (2022) 28:81–8. 10.1038/s41591-021-01650-w
166.
SharmaABoelensJJCancioMHankinsJSBhadPAzizyMet alCRISPR-Cas9 Editing of the HBG1 and HBG2 Promoters to Treat Sickle Cell Disease. N Engl J Med (2023) 389:820–32. 10.1056/NEJMoa2215643
167.
SouzaDCHebertNEsrickEBCiuculescuMFArcherNMArmantMet alGenetic Reversal of the Globin Switch Concurrently Modulates Both Fetal and Sickle Hemoglobin and Reduces Red Cell Sickling. Nat Commun (2023) 14:5850. 10.1038/s41467-023-40923-5
168.
EapenMBrazauskasRWilliamsDAWaltersMCSt MartinAJacobsBLet alSecondary Neoplasms After Hematopoietic Cell Transplant for Sickle Cell Disease. J Clin Oncol (2023) 41:2227–37. 10.1200/JCO.22.01203
169.
CarvalhoT. Discontinued CRISPR Gene Therapy for Sickle-Cell Disease Improves Symptoms. Nat Med (2023) 29(11):2669–70. 10.1038/d41591-023-00088-6
170.
NikitinDBeaudoinFLThokalaPMcKennaANhanERindDMet alGene Therapies for Sickle Cell Disease: Effectiveness and Value. J Manag Care Spec Pharm (2023) 29(11):1253–9. 10.18553/jmcp.2023.29.11.1253
171.
Spencer ChapmanMCullAHCiuculescuMFEsrickEBMitchellEJungHet alClonal Selection of Hematopoietic Stem Cells After Gene Therapy for Sickle Cell Disease. Nat Med (2023) 16:3175–83. 10.1038/s41591-023-02636-6
172.
NeelJVItanoHALawrenceJS. Two Cases of Sickle Cell Disease Presumably due to the Combination of the Genes for Thalassaemia and Sickle Cell Haemoglobin. Blood (1953) 8:434–43.
173.
AksoyM. The First Observation of Homozygous Hemoglobin S-Alpha Thalassemia Disease and Two Types of Sickle Cell Thalassemia Disease: (A) Sickle Cell-Alpha Thalassemia Disease, (B) Sickle cell-beta Thalassemia Disease. Blood (1963) 22:757–69.
174.
WeatherallDJCleggJBBlanksonJMcNeilJR. A New Sickling Disorder Resulting from Interaction of the Genes for Haemoglobin S and a-Thalassaemia. Br. J. Haematol (1969) 17:517–26. 10.1111/j.1365-2141.1969.tb01402.x
175.
SteinbergMH. Haemoglobin C/alpha Thalassaemia: Haematological and Biosynthetic Studies. Br J Haematol (1975) 30(3):337–42. 10.1111/j.1365-2141.1975.tb00549.x
176.
HiggsDRCleggJBWoodWGWeatherallDJ. G Gamma Beta + Type of Hereditary Persistence of Fetal Haemoglobin in Association with Hb C. J Med Genet (1979) 16(4):288–95. 10.1136/jmg.16.4.288
177.
WHO Working Group. Hereditary Anaemias: Genetic Basis, Clinical Features, Diagnosis, and Treatment. WHO Working Group. Bull World Health Organ (1982) 60(5):643–60.
178.
FucharoenSFucharoenGSanchaisuriyaKSurapotS. Compound Heterozygote States for Hb C/Hb Malay and Hb C/Hb E in Pregnancy: A Molecular and Hematological Analysis. Blood Cells Mol Dis (2005) 35(2):196–200. 10.1016/j.bcmd.2005.05.004
179.
PowarsDRChanLSchroederWA. Beta S-Gene-cluster Haplotypes in Sickle Cell Anemia: Clinical Implications. Am J Pediatr Hematol Oncol (1990) 12(3):367–74. 10.1097/00043426-199023000-00022
180.
MatthayKKMentzerWCJrDozyAMKanYWBaintonDF. Modification of Hemoglobin H Disease by Sickle Trait. J Clin Invest (1979) 64(4):1024–32. 10.1172/JCI109539
181.
KundrapuSJanakiNMeyersonHJ. Compound Heterozygosity for Hb D-Ibadan (HBB: C.263C>A) and Hb C (HBB: C.19G>A). Hemoglobin (2018) 42(4):269–71. 10.1080/03630269.2018.1523799
182.
WilcoxIBoettgerKGreeneLMalekADavisLSteinbergMHet alHemoglobin Kenya Composed of alpha- and ((A)gammabeta)-Fusion-Globin Chains, Associated with Hereditary Persistence of Fetal Hemoglobin. Am J Hematol (2009) 84(1):55–8. 10.1002/ajh.21308
183.
Redding-LallingerRTankutGHolleyLWrightFKutlarAKutlarF. Molecular Characterization of Hb D-Ibadan [beta87(F3)Thr--Lys] in Combination with Hb S [beta6(A3)Glu--Val] and with beta+-Thalassemia: Report of Two Cases. Hemoglobin (2002) 26(2):129–34. 10.1081/hem-120005450
184.
WallerPBlannAD. Non-Coding Rnas - a Primer for the Laboratory Scientist. Br J Biomed Sci (2019) 76(4):157–65. 10.1080/09674845.2019.1675847
185.
VacekMMMaHGemignaniFLacerraGKafriTKoleR. High-Level Expression of Hemoglobin A in Human Thalassemic Erythroid Progenitor Cells Following Lentiviral Vector Delivery of an Antisense snRNA. Blood (2003) 101(1):104–11. 10.1182/blood-2002-06-1869
186.
ChenSYWangYTelenMJChiJT. The Genomic Analysis of Erythrocyte microRNA Expression in Sickle Cell Diseases. PLoS One (2008) 3(6):e2360. 10.1371/journal.pone.0002360
187.
LulliVRomaniaPMorsilliOCianciulliPGabbianelliMTestaUet alMicroRNA-486-3p Regulates γ-globin Expression in Human Erythroid Cells by Directly Modulating BCL11A. PLoS One (2013) 8(4):e60436. 10.1371/journal.pone.0060436
188.
GasparelloJFabbriEBianchiNBreveglieriGZuccatoCBorgattiMet alBCL11A mRNA Targeting by miR-210: A Possible Network Regulating γ-Globin Gene Expression. Int J Mol Sci (2017) 18(12):2530. 10.3390/ijms18122530
189.
LeecharoenkiatKTanakaYHaradaYChaichompooPSarakulOAbeYet alPlasma microRNA-451 as a Novel Hemolytic Marker for β0-Thalassemia/HbE Disease. Mol Med Rep (2017) 15(5):2495–250. 10.3892/mmr.2017.6326
190.
LaiKJiaSYuSLuoJHeY. Genome-Wide Analysis of Aberrantly Expressed lncRNAs and miRNAs with Associated Co-Expression and ceRNA Networks in β-Thalassemia and Hereditary Persistence of Fetal Hemoglobin. Oncotarget (2017) 8(30):49931–43. 10.18632/oncotarget.18263
191.
CyrusC. The Role of miRNAs as Therapeutic Tools in Sickle Cell Disease. Medicina (2021) 57(10):1106. 10.3390/medicina57101106
192.
Starlard-DavenportAFitzgeraldAPaceBS. Exploring Epigenetic and microRNA Approaches for γ-Globin Gene Regulation. Exp Biol Med (2021) 246(22):2347–57. 10.1177/15353702211028195
193.
MasonPJ. New Insights into G6PD Deficiency. Br J Haematol (1996) 94:585–91.
194.
GuindoAFairhurstRMDoumboOKWellemsTEDialloDA. X-Linked G6PD Deficiency Protects Hemizygous Males but Not Heterozygous Females Against Severe Malaria. PLoS Med (2007) 4:e66. 10.1371/journal.pmed.004006659
195.
RuwendeCKhooSCSnowRWYatesSNKwiatkowskiDGuptaSet alNatural Selection of hemi- and Heterozygotes for G6PD Deficiency in Africa by Resistance to Severe Malaria. Nature (1995) 376:246–9. 10.1038/376246a0
196.
NkhomaETPooleCVannappagariVHallSABeutlerE. The Global Prevalence of Glucose-6-Phosphate Dehydrogenase Deficiency: A Systematic Review and Meta-Analysis. Blood Cells Mol Dis (2009) 42(3):267–78. 10.1016/j.bcmd.2008.12.005
197.
AlangariASEl-MetwallyAAAlanaziAAl KhateebBFAl KadriHMAlshdoukhiIFet alEpidemiology of Glucose-6-Phosphate Dehydrogenase Deficiency in Arab Countries: Insights from a Systematic Review. J Clin Med (2023) 12(20):6648. 10.3390/jcm12206648
198.
HowesREPielFBPatilAPNyangiriOAGethingPWDewiMet alG6PD Deficiency Prevalence and Estimates of Affected Populations in Malaria Endemic Countries: A Geostatistical Model-based Map. PLoS Med (2012) 9(11):e1001339. 10.1371/journal.pmed.1001339
199.
PersicoMCVigliettoGMartiniGTonioloDPaonessaGMoscatelliCet alIsolation of Human Glucose-6-Phosphate Dehydrogenase (G6PD) Cdna Clones: Primary Structure of the Protein and Unusual 5’ Non-Coding Region. Nucleic Acids Res (1986) 14:2511–22. 10.1093/nar/14.6.2511
200.
MartiniGTonioloDVulliamyTLuzzattoLDonoRVigliettoGet alStructural Analysis of the X-linked Gene Encoding Human Glucose 6-Phosphate Dehydrogenase. EMBO J (1986) 5:1849–55. 10.1002/j.1460-2075.1986.tb04436.x
201.
TakizawaTHuangIYIkutaTYoshidaA. Human Glucose-6-Phosphate Dehydrogenase: Primary Structure and cDNA Cloning. Proc Natl Acad Sci USA (1986) 83:4157–61. 10.1073/pnas.83.12.4157
202.
VulliamyTJD'UrsoMBattistuzziGEstradaMFoulkesNSMartiniGet alDiverse Point Mutations in the Human Glucose-6-Phosphate Dehydrogenase Gene Cause Enzyme Deficiency and Mild or Severe Hemolytic Anemia. Proc Natl Acad Sci U S A. (1988) 85(14):5171–5. 10.1073/pnas.85.14.5171
203.
BeutlerE. G6PD Deficiency. Blood (1994) 84(11):3613–36.
204.
SecrestMHStormMCarringtonCCassoDGilroyKPladsonLet alPrevalence of Pyruvate Kinase Deficiency: A Systematic Literature Review. Eur J Haematol (2020) 105:173–84. 10.1111/ejh.13424
205.
FoyPHigaSZhaoJKeapoletsweKCirneanuLVenerusAet alOverall Survival of Patients with Pyruvate Kinase Deficiency in the UK: A Real-World Study. eJHaem (2025) 6:e70009. 10.1002/jha2.70009
206.
PangYQiXQinJZhaiXWangRCaoJet alCase Report: Modified Transplantation for Pediatric Patients with Pyruvate Kinase Deficiency. Front Immunol (2024) 15:1493398. 10.3389/fimmu.2024.1493398
207.
OttoJC. An Account of an Hemorhagic Disposition Existing in Certain Families. Med Phys J (1808) 20(113):69–72.
208.
WalkerJW. On Haemophilia. Br Med J. (1872) 1(597):605–7. 10.1136/bmj.1.597.605
209.
Anonymous. Treatment of Haemophilia by Injections of Serum. Atlanta J Rec Med. (1907) 8(11):764–5.
210.
LingGTuddenhamEGD. Factor VIII: The Protein, Cloning Its Gene, Synthetic Factor and now – 35 Years Later – Gene Therapy; What Happened in Between?Br J Haematol (2020) 189:400–7. 10.1111/bjh.16311
211.
GitschierJWoodWIGoralkaTMWionKLChenEYEatonDHet alCharacterization of the Human Factor VIII Gene. Nature (1984) 312(5992):326–30. 10.1038/312326a0
212.
WoodWICaponDJSimonsenCCEatonDLGitschierJKeytBet alExpression of Active Human Factor VIII from Recombinant DNA Clones. Nature (1984) 312(5992):330–7. 10.1038/312330a0
213.
TooleJJKnopfJLWozneyJMSultzmanLABueckerJLPittmanDDet alMolecular Cloning of a cDNA Encoding Human Antihaemophilic Factor. Nature (1984) 312(5992):342–7. 10.1038/312342a0
214.
GitschierJLawnRMRotblatFGoldmanETuddenhamEG. Antenatal Diagnosis and Carrier Detection of Haemophilia A Using Factor VIII Gene Probe. Lancet (1985) 1(8437):1093–4. 10.1016/s0140-6736(85)92388-8
215.
AntonarakisSECopelandKLCarpenterRJJrCartaCAHoyerLWCaskeyCTet alPrenatal Diagnosis of Haemophilia A by Factor VIII Gene Analysis. Lancet (1985) 1(8443):1407–9. 10.1016/s0140-6736(85)91842-2
216.
DinNSchwartzMKruseTVestergaardSRAhrensPCaputDet alFactor VIII Gene Specific Probe for Prenatal Diagnosis of Haemophilia A. Lancet (1985) 1(8443):1446–7. 10.1016/s0140-6736(85)91873-2
217.
GitschierJWoodWITuddenhamEGShumanMAGoralkaTMChenEYet alDetection and Sequence of Mutations in the Factor VIII Gene of Haemophiliacs. Nature (1985) 315(6018):427–30. 10.1038/315427a0
218.
TantravahiUMurtyVVJhanwarSCTooleJJWoozneyJMChagantiRSet alPhysical Mapping of the Factor VIII Gene Proximal to Two Polymorphic DNA Probes in Human Chromosome Band Xq28: Implications for Factor VIII Gene Segregation Analysis. Cytogenet Cell Genet (1986) 42:75–9. 10.1159/000132255
219.
YoussoufianHWongCAronisSPlatokoukisHKazazianHHAntonarakisSE. Moderately Severe Hemophilia A Resulting from Glu----Gly Substitution in Exon 7 of the Factor VIII Gene. Am J Hum Genet (1988) 42(6):867–71.
220.
BardoniBSampietroMRomanoMCrapanzanoMMannucciPMCamerinoG. Characterization of a Partial Deletion of the Factor VIII Gene in a Haemophiliac with Inhibitor. Hum Genet (1988) 79:86–8. 10.1007/BF00291718
221.
YoussoufianHAntonarakisSEBellWGriffinAMKazazianHH. Nonsense and Missense Mutation in Hemophilia A: Estimate of the Relative Mutation Rate at CG Dinucleotides. Am J Hum Genet (1988) 42:718–25.
222.
AntonarakisSEKazazianHHJr. The Molecular Basis of Hemophilia A in Man. Trends Genet (1988) 4:233–7. 10.1016/0168-9525(88)90156-4
223.
WhiteGCShoemakerCB. Factor VIII Gene and Hemophilia A. Blood (1989) 73:1–12.
224.
NaylorJAGreenPMRizzaCRGiannelliF. Factor VIII Gene Explains all Cases of Haemophilia A. Lancet (1992) 340:1066–7. 10.1016/0140-6736(92)93080-7
225.
BiggsRDouglasASMacFarlaneRGDacieJVPitneyWRMerskeyC. Christmas Disease: A Condition Previously Mistaken for Haemophilia. Br Med J (1952) 2(4799):1378–82. 10.1136/bmj.2.4799.1378
226.
ChooKHGouldKGReesDJBrownleeGG. Molecular Cloning of the Gene for Human Anti-Haemophilic Factor IX. Nature (1982) 299:178–80. 10.1038/299178a0
227.
AnsonDSChooKHReesDJGiannelliFGouldKHuddlestonJAet alThe Gene Structure of Human Anti-Haemophilic Factor IX. EMBO J (1984) 3:1053–60. 10.1002/j.1460-2075.1984.tb01926.x
228.
McGrawRADavisLMLundbladRLStaffordDWRobertsHR. Structure and Function of Factor IX: Defects in Haemophilia B. Clin Haematol (1985) 14:359–83.
229.
KurachiKDavieEW. Isolation and Characterisation of a cDNA Coding for Human Factor IX. Proc Natl Acad Sci (1982) 79:6461–4. 10.1073/pnas.79.21.6461
230.
XuZSpencerHJHarrisVAPerkinsSJ. An Updated Interactive Database for 1692 Genetic Variants in Coagulation Factor IX Provides Detailed Insights into Hemophilia B. J Thromb Haemostas (2023) 21:1164–76. 10.1016/j.jtha.2023.02.005
231.
JohnsenJMFletcherSNHustonHRobergeSMartinBKKircherMet alNovel Approach to Genetic Analysis and Results in 3000 Hemophilia Patients Enrolled in the My Life, Our Future Initiative. Blood Adv (2017) 1:824–34. 10.1182/bloodadvances.2016002923
232.
HerzogRWKaczmarekRHighKA. Gene Therapy for Hemophilia - from Basic Science to First Approvals of “One-and-Done” Therapies. Mol Ther (2025) 33:2015–34. 10.1016/j.ymthe.2025.03.043
233.
PipeSWMiesbachWRechtMLeebeekFWGKeyNSCastamanGet alFinal Analysis of a Study of Etranacogene Dezaparvovec for Hemophilia B. N Engl J Med (2026) 394:463–74. 10.1056/NEJMoa2514332
234.
F8 coagulation factor VIII. Available online at: https://www.ncbi.nlm.nih.gov/gene/2157 (Accessed February 15th, 2026).
235.
DouglasTGSantosSHindsDRHatswellAJTaylorK. Comparative Effectiveness of Valoctocogene Roxaparvovec and Efanesoctocog Alfa in the Treatment of Severe Hemophilia A: A Matching-Adjusted Indirect Comparison of Bleeding Frequency. Adv Ther (2025) 42:5600–11. 10.1007/s12325-025-03339-9
236.
JacksonDP. Hereditary Disorders of Blood Coagulation due to Defective and Deficient Synthesis of Protein. Trans Am Clin Climatol Assoc (1971) 82:114–23.
237.
JennyRJPittmanDDTooleJJKrizRWAldapeRAHewickRMet alComplete cDNA and Derived Amino Acid Sequence of Human Factor V. Proc Natl Acad Sci U S A (1987) 84:4846–50. 10.1073/pnas.84.14.4846
238.
AsseltaRPeyvandiF. Factor V Deficiency. Semin Thromb Haemost (2009) 35:382–9. 10.1055/s-0029-1225760
239.
ZherngCZhangB. Combined Deficiency of Coagulation Factors V and VIII: An Update. Semin Thromb Hemost (2013) 39:613–20. 10.1055/s-0033-1349223
240.
BernardiFMarianiG. Biochemical, Molecular and Clinical Aspects of Coagulation Factor VII and Its Role in Hemostasis and Thrombosis. Haematologica (2021) 106:351–62. 10.3324/haematol.2020.248542
241.
PallaRPeyvandiFShapiroAD. Rare Bleeding Disorders: Diagnosis and Treatment. Blood (2015) 125:2052–61. 10.1182/blood-2014-08-532820
242.
TisciaGLMargaglioneM. Human Fibrinogen: Molecular and Genetic Aspects of Congenital Disorders. Int J Mol Sci (2018) 19:1597. 10.3390/ijms19061597
243.
MumfordADAckroydSAlikhanRBowlesLChowdaryPGraingerJet alGuideline for the Diagnosis and Management of the Rare Coagulation Disorders. Br J Haematol (2014) 167:304–26. 10.1111/bjh.13058
244.
RodeghieroFCastamanGDiniE. Epidemiological Investigation of the Prevalence of Von Willebrand’s Disease. Blood (1987) 69:454–9.
245.
BowmanMHopmanWMRapsonDLillicrapDJamesP. The Prevalence of Symptomatic Von Willebrand Disease in Primary Care Practice. J Thromb Haemostas (2010) 8:213–6. 10.1111/j.1538-7836.2009.03661.x
246.
GinsburgDHandinRIBonthronDTDonlonTABrunsGALattSAet alHuman Von Willebrand Factor (vWF): Isolation of Complementary DNA (cDNA) Clones and Chromosomal Localization. Science (1985) 228:1401–6. 10.1126/science.3874428
247.
ManderstedtELind-HalldenCLethagenSHalldénC. Genetic Variation in the Von Willebrand Factor Gene in Swedish Von Willebrand Disease Patients. TH Open (2018) 2(1):e39–e48. 10.1055/s-0037-1618571
248.
BlannAD. Plasma Von Willebrand Factor, Thrombosis, and the Endothelium: The First 30 Years. Thromb Haemost (2006) 95(1):49–55.
249.
AsmisLHellsternP. Thrombophilia Testing - a Systematic Review. Clin Lab (2023) 69. 10.7754/Clin.Lab.2022.220817
250.
MooreGW. Thrombophilia Screening: Not so Straightforward. Seminars Thromb Haemostas (2024) 50:1131–52. 10.1055/s-0044-1786807
251.
BlajchmanMAAustinRCFrenandex-RachubinskiFSheffieldWP. Molecular Basis of Inherited Human Antithrombin Deficiency. Blood (1992) 80:2159–71.
252.
Alhenc-GelasMPlu-BureauGHugon-RodinJPicardVHorellouMH. Thrombotic Risk According to SERPINC1 Genotype in a Large Cohort of Subjects with Antithrombin Inherited Deficiency, Thromb. Haemost (2017) 117:1040–51. 10.1160/TH16-08-0635
253.
ZollersBSvenssonPJHeXDahlbäckB. Identification of the Same Factor V Gene Mutation in 47 out of 50 Thrombosis-Prone Families with Inherited Resistance to Activated Protein C. J Clin Invest (1994) 94:2521–4. 10.1172/JCI117623
254.
KujovichJL. Factor V Leiden Thrombophilia. Genetics Med (2011) 13:1–16. 10.1097/GIM.0b013e3181faa0f2
255.
McGlennenRCKeyNS. Clinical and Laboratory Management of the Prothrombin G20210A Mutation. Arch Pathol Lab Med (2002) 126:1319–25. 10.5858/2002-126-1319-CALMOT
256.
ArachchillageDJMackillopLChandrathevaAMotawaniJMacCallumPLaffanM. Thrombophilia Testing: A British Society for Haematology Guideline. Br J Haematol (2022) 198:443–58. 10.1111/bjh.18239
257.
NurdenATNurdenP. Inherited Thrombocytopenias: History, Advances and Perspectives. Haematologica (2020) 105:2004–19. 10.3324/haematol.2019.233197
258.
LopezJAAndrewsRKAfshar-KharghanVBerndtMC. Bernard-Soulier syndrome. Blood (1998) 91:4397–418.
259.
LanzaF. Bernard-Soulier syndrome (Hemorrhagiparous Thrombocytic Dystrophy) Orphan. J Rare Dis (2006) 1:46. 10.1186/1750-1172-1-46
260.
NurdenA. Profiling the Genetic and Molecular Characteristics of Glanzmann Thrombasthenia: Can It Guide Current and Future Therapies?J Blood Med (2021) 12:581–99. 10.2147/JBM.S273053
261.
WarrenAJ. Molecular Basis of the Human Ribosomopathy Shwachman-Diamond syndrome. Adv Biol Regul (2018) 67:109–27. 10.1016/j.jbior.2017.09.002
262.
SeriMPecciADi BariFCusanoRSavinoMPanzaEet alMYH9-Related Disease: May-Hegglin Anomaly, Sebastian Syndrome, Fechtner syndrome, and Epstein Syndrome Are Not Distinct Entities but Represent a Variable Expression of a Single Illness. Medicine (2003) 82:203–15. 10.1097/01.md.0000076006.64510.5c
263.
HsuAP. Not Too Little, Not Too Much: The Impact of Mutation Types in Wiskott-Aldrich syndrome and RAC2 Patients. Clin Exp Immunol (2023) 212:137–46. 10.1093/cei/uxad001
264.
AlbersCACvejicCFavierRBouwmansEEAlessiMCBertonePet alExome Sequencing Identifies NBEAL2 as the Causative Gene for Gray Platelet Syndrome. Nat Genet (2012) 43:735–7. 10.1038/ng.885
265.
BottegaRNicchiaEAlfanoCGlembotskyACPastoreABertaggia-CalderaraDet alGray Platelet Syndrome: Novel Mutations of the NBEAL2 Gene. Am J Hematol (2017) 92:E20–E22. 10.1002/ajh.24610
266.
GebetsbergerJMottKBernarAKlopockiEStreifWSchulzeH. State of the Art Targeted High-Throughput Sequencing for Detecting Inherited Platelet Disorders. Hamostaseologie (2023) 43:244–25. 10.1055/a-2099-3266
267.
KalfaTA. Diagnosis and Clinical Management of Red Cell Membrane Disorders. Hematology Am Soc Hematol Educ Program (2021) 2021:331–40. 10.1182/hematology.2021000265
268.
FattizzoBGiannottaJACecchiNBarcelliniW. Confounding Factors in the Diagnosis and Clinical Course of Rare Congenital Hemolytic Anemias. Orphanet J Rare Dis (2021) 16:415. 10.1186/s13023-021-02036-4
269.
MohandasN. Inherited Hemolytic Anemia: A Possessive Beginner's Guide. Hematology Am Soc Hematol Educ Program (2018) 2018:377–81. 10.1182/asheducation-2018.1.377
270.
IolasconAAndolfoIRussoR. Advances in Understanding the Pathogenesis of Red Cell Membrane Disorders. Br J Haematol (2019) 187:13–24. 10.1111/bjh.16126
271.
ZamaDGiuliettiGMuratoreEAndolfoIRussoRIolasconAet alA Novel PIEZO1 Mutation in a Patient with Dehydrated Hereditary Stomatocytosis: A Case Report and a Brief Review of Literature. Ital J Pediatr (2020) 46(1):102. 10.1186/s13052-020-00864-x
272.
AndolfoIRussoRGambaleAIolasconA. Hereditary Stomatocytosis: An Underdiagnosed Condition. Amer J Haematol (2018) 93:107–21. 10.1002/ajh.24929
273.
PietrangeloA. Hereditary Hemochromatosis. Biochim Biophys Acta (2006) 1763:700–10. 10.1016/j.bbamcr.2006.05.013
274.
PillingLCTamosauskaiteJJonesGWoodARJonesLKuoCLet alCommon Conditions Associated with Hereditary Haemochromatosis Genetic Variants: Cohort Study in UK Biobank. BMJ (2019) 364:k5222. 10.1136/bmj.k5222
275.
AlvarengaAMBrissotPSantosPCJL. Haemochromatosis Revisited. World J Hepatol (2022) 14:1931–9. 10.4254/wjh.v14.i11.1931
276.
KowdleyDKowdleyKV. Appropriate Clinical Genetic Testing of Hemochromatosis Type 2-4, Including Ferroportin Disease. Applics Clin Genetics (2021) 14:353–61. 10.2147/TACG.S269622
277.
PietrangeloA. Ferroportin Disease: Pathogenesis, Diagnosis and Treatment. Haematologica (2017) 102:1972–84. 10.3324/haematol.2017.170720
278.
BellSRigasASMagnussonMKFerkingstadEAllaraEBjornsdottirGet alA Genome-Wide Meta-Analysis Yields 46 New Loci Associating with Biomarkers of Iron Homeostasis. Commun Biol (2021) 4:156. 10.1038/s42003-020-01575-z
279.
RoettoATotaroAPipernoAPigaALongoFGarozzoGet alNew Mutations Inactivating Transferrin Receptor 2 in Hemochromatosis Type 3. Blood (2001) 97(9):2555–60. 10.1182/blood.V97.9.2555
280.
WangBWangXP. Does Ceruloplasmin Defend Against Neurodegenerative Diseases?Curr Neuropharmacol (2019) 17(6):539–49. 10.2174/1570159X16666180508113025
281.
McCaulNBinghamRJBlannAD. The Molecular Pathology of Blood Cancer: A Narrative Review. Br J Biomed Sci 2026: Details to Be Provided by Frontiers in Due Course – You’d Best Refer to Them82:14745. 10.3389/bjbs.2025.14745
Summary
Keywords
coagulopathy, molecular pathology, haematology, haemoglobinopathy, molecular genetics
Citation
Blann AD and Dunn RG (2026) The Molecular Pathology of Non-Malignant Haematological Disease. Br. J. Biomed. Sci. 83:14743. doi: 10.3389/bjbs.2026.14743
Received
09 April 2025
Revised
12 November 2025
Accepted
17 February 2026
Published
22 May 2026
Volume
83 - 2026
Updates
Copyright
© 2026 Blann and Dunn.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: A. D. Blann, andrew.blann@hotmail.com
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.