Large β Globin Gene Cluster Deletions and Implications on Transcription Factor Regulation in Hemoglobin Switching - Α Novel ε Gγ Αγ Deletion with Intact LCR

The εγδβ thalassemias (EGDBT) are infrequently occurring deletions of the β globin gene cluster on chromosome 11. EGDBTs can be an unsuspected cause of severe neonatal anemia that resolves during the first year of life into a normal Hb A2 thalassemic phenotype, presumed to be due to an imbalance of β-like chains during development. First described in 1972, they are categorized into two groups, both with loss of the five DNase I hypersensitivity sites upstream of the β gene (HBB) called the locus control region (LCR). In group 1 deletions, all or most of the β globin gene cluster including HBB is deleted; in group 2 deletions, removal of the LCR silences an intact HBB. Large deletions that do not involve LCR but remove the embryonic (HBE) and fetal genes (HBG2/HBG1) [LCR(ε Gγ Aγ) 0δβ] have not been reported. It is not known whether a deletion with intact LCR as well as δ and β genes would similarly manifest as neonatal anemia that resolves into a normocytic phenotype with normal hemoglobin fraction percentages after infancy. Four individuals with an LCR(ε Gγ Aγ) 0δβ deletion have been evaluated. These include monochorionic, diamniotic twin girls whose newborn screen (NBS) returned significant for Hb A percentage greater than Hb F for both twins. NBS was repeated two weeks later with the same result. The newborns were induced late pre-term (36 weeks and 6 days) after an uncomplicated pregnancy, including no features of twin to twin transfusion. Both infants had no anemia and bilirubin levels remained in the low to low-intermediate risk zones. Neither twin received transfusions or phototherapy during admission. At a follow up visit at 20 months of age a hemoglobin electrophoresis was performed which showed borderline Hb A2 and no Hb F in both twins (Table 1). Ferritin levels were low-normal in Twin A (14 µg/L) and low in Twin B (9 µg/L). Both infants were found to have a heterozygous large deletion affecting HBE, HBG2, HBG1, and HBBP1 loci by Multiplex Ligation-dependent Probe Amplification (MLPA), a method that uses multiple DNA probe pairs to determine the size of the DNA segment deleted. MLPA deletion/duplication testing on the α genes was negative. No β globin variants were identified by DNA sequencing. An evaluation was performed on the 25 year-old healthy mother and showed similar findings including the heterozygous large deletion. Mother and infants continue to be asymptomatic with normal red cell indices for age (Table 1). The fourth was an unrelated 40-year-old female with mild anemia screened by hemoglobin electrophoresis for obstetric evaluation who was found to have borderline Hb A2 (see table 1). MLPA showed a heterozygous deletion involving the same loci. Again, no β globin variants were identified by DNA sequencing. All four cases showed the same MLPA pattern except one non-specific probe commonly altered in common gamma gene conversion events. Long range sequencing performed on one case confirmed a single contiguous 32,599 bp deletion that matched the MLPA data (g.5262276-5294875). The detected deletion has not been reported in literature and differs from EGDBT mutations in that the LCR is intact. As the LCR controls expression of the entire gene cluster, this mutation is expected to display different phenotypic features than classic EGDBT. HBD and HBB are not deleted and therefore adequate transcription of Hb A2 and Hb A are expected with no imbalance of α/β chains after the neonatal period. It is postulated this finding explains the decreased Hb F levels in the NBS test result. The Hb A2 increase is likely a product of this deletion and does not indicate β-thalassemia. The heterozygous absence of HBE and HBG2/HBG1 may result in upregulation of HBD and HBB expression through loss of FKLF and FKLF-2 binding. In addition, HBB-EKLF binding would have less competition as the gamma promoter elements have been heterozygously deleted. In summary, in contrast to classic EGDBTs which cause transient severe neonatal anemia and resolve to normal Hb A2 thalassemic indices, this novel LCR(ε Gγ Aγ) 0δβ deletion was associated with normal CBC values with an absence of severe neonatal anemia, inverted Hb F/Hb A percentages at birth and borderline Hb A2 levels. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Characterization of β-Globin Gene Cluster Deletions Using Multiplex-Gap Polymerase Chain Reaction (PCR) and Multiplex Ligation-Dependent Probe Amplification (MLPA)

Background : Deletions in the β-globin gene cluster are usually rare, problematic to detect, and subsequently possess a challenge in many diagnostic laboratories. They are normally related to the heterozygous of the delta beta (δβ) thalassemia, hereditary persistence of fetalhemoglobin (HPFH) and some of the hemoglobin variants. These disorders are typically presented by elevated levels of hemoglobin F (Hb F), but with low to normal hemoglobin A2 (Hb A2). However, despite their existence, there is still a limited number of studies focusing on this area, hence no definitive diagnosis could be conclusively established. Therefore, this pilot study was carried out to fill this knowledge gap. Methods: In this study, screening of the selected deletional mutations in the β-globin gene cluster among patients with Hb F (>1%) and Hb A2 (<4%) were performed using multiplex Gap-PCR and multiplex ligation-dependent probe amplification (MLPA). Results: The results showed that out of 54 samples tested using multiplex Gap-PCR against four target deletions; Thai (δβ)°-thalassemia, HPFH-6, Siriraj J and HbLepore, one sample was found positive with Thai (δβ)°-thalassemia. Further findings from the MLPA screening on 12 randomly selected samples revealed that another patient was positive with double deletions within the region of the β-globin gene cluster. These deletions occur at the gamma-globin gene 1 (HBG1) and gamma-globin gene 2 (HBG2) in exon 3. Conclusions: In conclusion, this study highlighted the importance of these deletions’ characterization using multiplex Gap-PCR and MLPA which helps in establishing a definitive diagnosis among this selected group of patients. Bangladesh Journal of Medical Science Vol.20(3) 2021 p.618-624

β-globin gene cluster mutations and large deletions among anaemic patients with elevated HbF level in a tertiary teaching hospital in Kelantan, Malaysia

Mutations in the β-globin gene cluster can lead to β-thalassaemia, δβ-thalassaemia, hereditary persistence of foetal haemoglobin (HPFH) and some of the haemoglobin variants. The clinical and haematological spectrum of thalassaemia range from benign to pathogenic conditions including severe transfusion dependent thalassaemia. Therefore, it is important to accurately diagnose β-globin gene cluster mutations to prevent thalassaemia major or intermedia offspring. The objective of this study is to detect β-globin gene cluster mutations and large deletions among anaemic patients with elevated HbF level in a tertiary teaching hospital in Kelantan, Malaysia. This study involved 144 anaemic patients with HbF level >1.0%. High-Performance Liquid Chromatography (HPLC) was used to determine the HbF and HbA2 levels. Multiplex-ARMS (ARMS)-PCR and gap-PCR were performed for those patients with high HbA2 level (>3.2%) and normal HbA2 level (≤3.2%) to detect β-globin gene cluster mutations and large deletions respectively. The majority of patients were Malays (99.3%) with mean age of 19.99 ± 1.64 years and female 61.1% predominance. Out of 87 samples tested using multiplex ARMS-PCR against eight targeted single mutation; Cd 41/42, IVS 1–5, Cd 26, Cd 17, Cd 71/72, IVS 1–1, Cd 8/9 and -28 mutations, 65 (74.7%) patients were detected which comprises of Cd 26 (56.3%), Cd 41/42 (11.5%), compound Cd 26 and Cd 41/42 (3.4%) and IVS 1–1 (3.4%). Meanwhile, for multiplex gap-PCR which detect four types of large deletions; Thai (δβ)o-thalassaemia, HPFH-6, Siriraj J and Hb Lepore, one out of 57 patients (1.8%) was found positive with Thai (δβ)o-thalassaemia. There was a significant difference between the mean of HbF level, MCV level as well as MCH level of patients with and without β-globin gene mutations and large deletions (P<0.05). This study highlighted the presence of various types of β-globin gene cluster mutations detection in establishing a definitive diagnosis among this selected group of patients for the large-scale screening of the thalassaemia gene.

Establishment and application of a novel method based on single nucleotide polymorphism analysis for detecting β-globin gene cluster deletions

β-Globin gene mutations reduce or terminate the production of beta globin chains, of which approximately 10% are large deletions within the β-globin gene cluster. Because gene deletion leads to loss of heterozygosity at single nucleotide polymorphism (SNP), a novel method for detecting β-globin gene cluster deletions based on SNP heterozygosity analysis was established in this study. The location range of SNPs was selected according to the breakpoint of β-globin gene cluster deletions. SNPs were screened using bioinformatics analysis and population sequencing data. A novel method which enables genotyping of multiplex SNPs based on tetra-primer ARMS-PCR was designed and optimized. Forty clinical samples were tested in parallel by this method and MLPA to verify the performance of this method for detecting β-globin gene cluster deletion. Six informative SNPs were obtained, achieving heterozygote coverage of 93.3% in normal individuals. Genotyping of six SNPs were successfully integrated into two multiplex tetra-primer ARMS-PCR reactions. The sensitivity, specificity, positive predictive value and negative predictive value of the method for detecting β-globin gene cluster deletion were 100%, 96.30%, 92.86%, and 100%, respectively. This is a simple, cost-effective and novel method for detecting β-globin gene cluster deletions, which may be suitable for use in combination with MLPA for thalassemia molecular testing.