β-Thalassemia is brought about by defective β-globin (HBB hemoglobin subunit β) formation and, in severe cases, requires regular blood transfusion and iron chelation for survival. Genome editing of ...hematopoietic stem cells allows correction of underlying mutations as curative therapy. As potentially safer alternatives to double-strand-break–based editors, base editors (BEs) catalyze base transitions for precision editing of DNA target sites, prompting us to reclone and evaluate two recently published adenine BEs (ABEs; SpRY and SpG) with relaxed protospacer adjacent motif requirements for their ability to correct the common HBBIVSI-110(G>A) splice mutation. Nucleofection of ABE components as RNA into patient-derived CD34+ cells achieved up to 90% editing of upstream sequence elements critical for aberrant splicing, allowing full characterization of the on-target base-editing profile of each ABE and the detection of differences in on-target insertions and deletions. In addition, this study identifies opposing effects on splice correction for two neighboring context bases, establishes the frequency distribution of multiple BE editing events in the editing window, and shows high-efficiency functional correction of HBBIVSI-110(G>A) for our ABEs, including at the levels of RNA, protein, and erythroid differentiation.
Display omitted
Lederer and colleagues have targeted HBBIVSI-110(G>A) thalassemia with novel base editors and shown that editing of an upstream base results in phenotypic correction. Alternatively, combinatorial base editing of three HBBIVSI-110(G>A)-proximal upstream bases has similarly high correction efficiency. By contrast, editing of an interspersed base is instead detrimental to normal splicing.
β thalassemia carriers are usually symptom free with microcytic hypochromic red cells and a raised HbA2 level. However, an increased output of α globin through co-inheritance of extra α globin genes, ...converts a typically asymptomatic β thalassemia carrier state to that of thalassemia intermedia. We describe 3 families with 3 unique head-to-tail duplications of the a-globin cluster in which all the probands have thalassemia intermedia ranging from moderately severe anemia with splenomegaly, to transfusion-dependence.
In Family 1, both father (Chinese) and son (Chinese and Anglo-Saxon English) were heterozygous for the HBB:c.316-197C>T β thalassemia variant but had moderately severe anemia (Hb 67 to 91 g/L) with splenomegaly; they were both transfusion-independent. In Family 2, the father (Syrian) had normal hematology, while mother (Iraqi) had a hematologic profile (Hb 110 g/L, RBC 5.68x1012/L, MCH 19.4 pg, MCV 58.9 fL and HbA2 4.8%) typical of heterozygous β0 thalassemia. The proband presented at age 5 years with severe anemia precipitated by an infection, that needed a blood transfusion. She continued to need intermittent blood transfusion while an older sister with a hematologic profile of Hb 75 g/L, RBC 3.04x1012/L, MCH 19.6 pg, MCV 65.6 fL, and mild spenomegaly, remained transfusion-independent. In Family 3 (Greek Cypriot), both parents were asymptomatic; the father was heterozygous for the HBB:c.93-21G>A β thalassemia variant with a normal a globin genotype (aa/aa), and the mother had normal HbA2 levels. In contrast, both their daughter and son who had inherited father's β thalassemia, had moderately severe anemia and needed intermittent blood transfusion. In all 3 families, MLPA suggested duplication of the whole alpha globin cluster but could not differentiate the different duplications.
Next generation sequencing using Agilent SureSelect bait capture, targeted sequence analysis to the two globin loci. Sequence alignment to the reference sequence was performed using NextGene (SoftGenetits, USA). Analysis of the β globin gene sequence identified the β thalassemia-causing variant in each family. Comparison of the sequence coverage across the a loci on chromosome 16 between each case and normal controls, showed that where duplications had occurred, there was proportionally more sequence captured, similar to SNP or CGH array analysis. At the point where the sequence coverage increased, a duplication breakpoint was suspected, and the aligned sequence reads were examined in detail. In Family 1, individual sequence reads matched part of the reference sequence, BLAT query in UCSC showed that the two halves of the read aligned at either end of the duplication, indicating they were sequences that spanned the head-to-tail breakpoint. This was confirmed by gap-PCR and Sanger sequence analysis. In the other two families, breakpoints were identified within repetitive regions which could not be captured by the baits and were therefore not covered by the captured sequence. The high resolution of the coverage map allowed precise characterisation of the duplications by gap-PCR and Sanger sequencing analysis of the breakpoint amplicons.
Duplication of the a globin cluster was encompassed in 188.7 kb in family 1, 120.5 kb in family 2, and 274 kb in family 3 (Figure 1). Both father and son in Family 1 were heterozygous for the duplicated a globin cluster (aa/aa, aa) and HBB:c.316-197C>T mutation. Both siblings in Family 2 were heterozygous for mother's β thalassemia c.135delC variant and father's duplicated a globin cluster. In family 3, the mother had a 3.7 kb a deletion variant and a duplicated a globin cluster (-a3.7/aa, aa), a total of 5 a globin genes. Both the daughter and son had inherited mother's duplicated a globin cluster with father's β thalassemia variant.
These families clearly show that a duplicated a globin cluster does not have a discernible phenotype on its own but is readily detectable when co-inherited with a β thalassemia variant. In all 3 cases, the a globin cluster duplications are in a head-to-tail orientation and occurred in repeats. These have most likely formed by non-homologous recombination events involving repetitive Alu sequences interspersed throughout the region. Whether these events occur more commonly in this region or if the region tolerates these changes better, allowing them to accumulate, remains to be resolved.
Display omitted
No relevant conflicts of interest to declare.
The detection and diagnosis of β-thalassaemia for populations with molecular heterogeneity, or diverse ethnic groups, has increased the need for the development of an array high-throughput diagnostic ...tool that can deliver large scale genetic detection. We report on the update and validation of the ThalassoChip, a β-thalassaemia genetic diagnostic tool which is based on arrayed primer extension (APEX) technology.
ThalassoChip slides with new and redesigned probes were prepared for testing the microarray. Six hundred and sixty DNA samples collected from eight Mediterranean countries were used for standardisation, optimisation and validation of the ThalassoChip. The β-globin gene region was amplified by PCR, the products were hybridised to the probes after fragmentation and the APEX reaction followed.
The ThalassoChip was updated with new probes and now has the ability to detect 57 β-globin gene mutations and three single nucleotide polymorphisms (SNPs) in a single test. The ThalassoChip as well as the PCR and APEX reactions were standardised and optimised using 500 DNA samples that were previously genotyped using conventional diagnostic techniques. Some probes were redesigned in order to improve the specificity and sensitivity of the test. Validation of the ThalassoChip performed using 160 samples analysed in blinded fashion showed no error.
The updated version of the ThalassoChip is versatile, robust, cost-effective and easily adaptable, but most notably can provide comprehensive genetic diagnosis for β-thalassaemia and other haemoglobinopathies.
Cyprus, located at the eastern end of the Mediterranean region, has been a place of eastern and western civilizations, and the presence of various hemoglobin (Hb) variants can be considered a ...testimony to past colonizations of the island. In this study, we report the structural Hb variants identified in the Cypriot population (Greek Cypriots, Maronites, Armenians, and Latinos) during the thalassemia screening of 248,000 subjects carried out at the Thalassaemia Centre, Nicosia, Cyprus, over a period of 26 years. A sample population of 65,668 people was used to determine the frequency and localization of several of the variants identified in Cyprus. The localization of some of the variants in regions where the presence of foreign people was most prevalent provides important clues to the origin of the variants. Twelve structural variants have been identified by DNA sequencing, nine concerning the β-globin gene and three concerning the α-globin gene. The most common β-globin variants identified were Hb S (0.2%), Hb D-Punjab (0.02%), and Hb Lepore-Washington-Boston (Hb Lepore-WB) (0.03%); the most common α-globin variant was Hb Setif (0.1%). The presence of some of these variants is likely to be directly linked to the history of Cyprus, as archeological monuments have been found throughout the island which signify the presence for many years of the Greeks, Syrians, Persians, Arabs, Byzantines, Franks, Venetians, and Turks.
We report two cases of compound heterozygote patients for the --(MED I) and Hb Agrinio alpha29(B10)Le-->uPro (alpha2) anomalies in two unrelated Greek Cypriot families. The first patient had a ...serious form of Hb H disease and died at the age of 21 due to complications arising during an operation. The second patient showed a severe hematological picture and has been regularly transfused since an early age. This patient exhibits bone abnormalities as well as hepatosplenomegaly. The severity of these two incidences emphasizes the need for the inclusion of a screening test for the --(MED I)/alpha(Agrinio)alpha genotype among those already offered during prenatal diagnosis. Two homozygotes, as well as a number of simple, compound, and double heterozygotes for Hb Agrinio have been identified in Cyprus and their hematological indices are presented.
We report two cases of compound heterozygote patients for the - -MED I and Hb Agrinio α29(B10)Leu→Pro (α2) anomalies in two unrelated Greek Cypriot families. The first patient had a serious form of ...Hb H disease and died at the age of 21 due to complications arising during an operation. The second patient showed a severe hematological picture and has been regularly transfused since an early age. This patient exhibits bone abnormalities as well as hepatosplenomegaly. The severity of these two incidences emphasizes the need for the inclusion of a screening test for the - -MED I αAgrinioα genotype among those already offered during prenatal diagnosis. Two homozygotes, as well as a number of simple, compound, and double heterozygotes for Hb Agrinio have been identified in Cyprus and their hematological indices are presented.
