How Molecular Pathology Rewrote the Story of Inherited Blood Disorders

Molecular pathology has transformed almost every corner of biomedical science, but few fields have felt its impact as profoundly as haematology. A new narrative review published in the British Journal of Biomedical Science traces how the discipline moved from the first isolation of DNA from leucocyte nuclei by Friedrich Miescher to CRISPR-based gene therapies now reshaping the treatment of sickle cell disease and thalassaemia. The review, which takes a deliberately historical perspective, argues that non-malignant blood disorders were not merely beneficiaries of the molecular revolution; they were its proving ground, where the tools of restriction enzymes, Southern blotting, polymerase chain reaction and next-generation sequencing were first sharpened against human disease.

The epidemiological case for this focus is striking. The Global Burden of Disease study recorded 55.9 million deaths in 2017, of which 834,700 were attributable to blood cancers and 104,600 to haemoglobinopathies and haemolytic anaemias. Yet prevalence tells a very different story: while the major blood cancers affect close to four million people worldwide, haemoglobinopathies and haemolytic anaemias affect roughly two billion, a five-hundred-fold difference. Years lived with disability follow the same pattern, with 6,572 million YLDs linked to haemoglobinopathies and haemolytic anaemias in 2016 compared with around 460,000 for blood cancer. The World Health Organization estimated in 2011 that around seven percent of the global population carries a potentially pathological haemoglobin gene, and more recent figures suggest three million people live with sickle cell disease alone, with more than 300,000 affected births each year.

The thalassaemias were the first disorders to yield their secrets to molecular genetics. During the 1960s and early 1970s, haemoglobin electrophoresis was the gold standard for studying abnormal globin chains, but by the mid-1970s restriction nucleases and Southern blotting were pinning down the aetiology at the level of DNA itself. Researchers demonstrated undetectable alpha-globin mRNA and deletions in the alpha-globin gene as causes of alpha-thalassaemia, and landmark work on beta-thalassaemia used reticulocyte mRNA purified on oligo-thymine columns, converted to cDNA with an RNA-dependent DNA polymerase from avian myeloblastosis virus, to show that affected patients produced alpha- and gamma-globin but no beta-globin. Orkin and colleagues later used extensive restriction endonuclease digestion to reveal partial and complete deletions of the beta-globin gene, while other studies established that three of the four alpha-globin genes are deleted in HbH disease and all four in Hb Bart’s foetal hydrops.

By the end of the 1970s the globin gene map was essentially complete: the alpha-globin locus, with its two copies HBA1 and HBA2, sits on chromosome 16 at 16p13.3, while the beta-globin gene HBB and the gamma-globin locus occupy chromosome 11 at 11p15.4. Sanger sequencing then exposed the precise nucleotide lesions, including G to T and T to G transversions and an A to G transition within beta-globin introns, all causing beta-thalassaemia, and a single base mutation at alpha-globin codon 116 converting GAG to a premature termination codon. Crucially, Baralle and colleagues sequenced 2,000 nucleotides upstream of the epsilon-globin gene, uncovering a regulatory promoter region and proving for the first time that a lesion outside an apparently intact gene could still cause a haemoglobinopathy. The 1990s added the locus control region, some 34 kilobases upstream of the epsilon gene, and the transcription factor KLF1, which binds CACCC boxes in promoter regions; a large deletion within the LCR produces gamma-delta-beta thalassaemia.

Next-generation sequencing has since deepened the picture, revealing alpha-gene duplications and quadruplications, refining prenatal diagnosis and enabling population screening. In Sardinia, where the beta-thalassaemia carrier rate is around twelve percent among 1.7 million people, modern platforms find that a quarter of at-risk foetuses are unaffected, half are healthy carriers and a quarter are affected, with over 98 percent of affected pregnancies terminated. Droplet digital PCR now improves copy number variation quantification, whole-exome sequencing exposes aberrant gene activity, and third-generation single-molecule platforms such as Pac-Bio, nanoball technology and Oxford Nanopore can resolve rare and complex thalassaemia variants that defeat short-read sequencing, offering a single test that captures both single nucleotide variants and copy number changes.

Gene therapy has moved from concept to clinic. CRISPR, linked with Cas9 endonuclease, earned Doudna and Charpentier the 2020 Nobel Prize, and one of its first practical applications was correcting a mutated beta-globin gene in an induced pluripotent stem cell line in vitro. A genome-wide association study of 4,305 Sardinians showed that BCL11A, coded at 2p16.1, strongly influences foetal haemoglobin levels, raising the hypothesis that suppressing this gene could reactivate HbF production in adults. In the landmark trial, patients with transfusion-dependent beta-thalassaemia and sickle cell disease received autologous CRISPR-Cas9-edited CD34-positive stem cells targeting BCL11A; haemoglobin rose from 90 to 141 g/L and from 72 to 120 g/L respectively, with parallel improvements in quality of life. A similar study editing the BCL11A enhancer lifted haemoglobin from 82 to 150 g/L and from 108 to 140 g/L, although a meta-analysis suggests the HBG1/2 promoter regions may be an even more efficient target.

The sickling disorders carry an even heavier global load. Sickle cell disease prevalence exceeds that of thalassaemia more than ten-fold, and by 2021 an estimated 515,000 babies were born with the condition each year, with 7.74 million people living with it. The leading evolutionary explanation, proposed seventy years ago, is that the mutation protects against Plasmodium falciparum malaria. Molecularly, the lesion is exquisitely simple: Marotta and colleagues purified mRNA from sickle reticulocytes, generated and sequenced cDNA by the Maxam-Gilbert method, and showed that HbS results from a single nucleotide change in the beta-globin gene converting the glutamic acid codon GAG at amino acid position 6 to the valine codon GTG. Subsequent decades brought allele-specific PCR, heteroduplex screening, and the demonstration that HbS polymerisation requires interaction between alpha-globin proline 114 and beta-globin threonine 87, knowledge that directly informed the design of antisickling gene variants.

Clinical gene therapy for sickle cell disease has accelerated rapidly. In 2017, Ribeil and colleagues transplanted a thirteen-year-old boy with his own CD34-positive stem cells engineered via lentiviral vector to carry the antisickling HBB variant beta-A-T87Q; fifteen months later his total haemoglobin had risen from 101 to 118 g/L with no recurrence of sickle crises. Esrick and colleagues silenced BCL11A with a short-hairpin RNA in six patients, raising HbF from 9.0 to 18.6 picograms per cell with robust and stable induction. The largest study to date, involving 35 patients receiving an engineered antisickling haemoglobin, saw median haemoglobin climb from 85 to over 120 g/L after 36 months, with no severe vaso-occlusive events after transplantation. Yet caution remains warranted: despite clinical improvement in the OTQ923 study targeting BCL11A, patients continued to show mild haemolysis, contributing to the sponsor’s decision to discontinue that product, and researchers have documented an increased frequency of potential driver mutations associated with myeloid neoplasms in both modified and unmodified stem cells after transplantation, alongside treatment costs approaching two million dollars per patient.

Beyond haemoglobin, the review surveys the enzymopathies, led by glucose-6-phosphate dehydrogenase deficiency, now affecting almost 1.2 billion people worldwide yet causing relatively few deaths because of its generally benign course and the variable penetrance of mutations in its Xq28 gene. Pyruvate kinase deficiency, the second most important enzymopathy, arises from more than 300 loss-of-function mutations in PKLR at 1q22 and shortens lifespan by roughly a decade. In haemostasis, the cloning of the Factor VIII gene in 1984, with its 186 kilobases, 25 introns and 26 exons, opened the era of molecular haemophilia diagnosis, and next-generation sequencing of 3,000 patients confirmed the intron 22 inversion as the leading mutation in severe haemophilia A. The compact Factor IX gene, at roughly 34 kilobases, fits readily into viral vectors for hepatocyte delivery, and the hyperactive FIX-Padua variant plus two approved haemophilia A therapeutics, valoctocogene roxaparvovec and efanesoctocog alfa, illustrate how molecular insight becomes clinical product. Von Willebrand disease, the most common genetic haemorrhagic disorder, stems from mutations in the 176-kilobase, 52-exon VWF gene, while thrombophilia panels now interrogate SERPINC1, Factor V Leiden, prothrombin G20210A, PROC and PROS1 in a single assay.

The review closes with the red cell membrane and iron metabolism. Hereditary spherocytosis, the most common hereditary haemolytic anaemia worldwide, is driven most often by mutations in ANK1 coding ankyrin, while hereditary elliptocytosis in West Africa traces to SPTA1, SPTB, SLC4A1 and EPB41, and Southeast Asian ovalocytosis, caused by a 27-nucleotide deletion in SLC4A1, confers resistance to cerebral malaria. Hereditary haemochromatosis, present homozygously in 0.2 to 0.33 percent of Europeans, arises overwhelmingly from the C282Y mutation in HFE, which disrupts hepcidin synthesis and permits unchecked tissue iron accumulation, with ferroportin disease and rarer HJV, HAMP and TFR2 variants completing the picture. The authors conclude that genetic testing will continue to expand across clinical and biomedical science, potentially reaching near-patient testing in primary care and even self-testing, allowing earlier disease recognition and more focused management with fewer complex referrals.

Subject of Research: The molecular pathology and gene therapy of inherited non-malignant haematological diseases

Article Title: The Molecular Pathology of Non-Malignant Haematological Disease

Article References: Blann, A. D., & Dunn, R. G. (2026). The Molecular Pathology of Non-Malignant Haematological Disease. British Journal of Biomedical Science, 83, Article 14743. https://doi.org/10.3389/bjbs.2026.14743

Image Credits: AI Generated

DOI: 10.3389/bjbs.2026.14743

Keywords: molecular pathology, haematology, thalassaemia, sickle cell disease, CRISPR, gene therapy, G6PD deficiency, haemophilia, von Willebrand disease, hereditary haemochromatosis, next-generation sequencing, non-coding RNA