Context base editing for splice correction of IVSI-110 ß-thalassemia.

ß-Thalassemia is brought about by defective ß-globin (HBB [hemoglobin subunit ß]) 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.

ThalassoChip, an array mutation and single nucleotide polymorphism detection tool for the diagnosis of ß-thalassaemia.

BACKGROUND: The detection and diagnosis of ß-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 ß-thalassaemia genetic diagnostic tool which is based on arrayed primer extension (APEX) technology. METHODS: 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 ß-globin gene region was amplified by PCR, the products were hybridised to the probes after fragmentation and the APEX reaction followed. RESULTS: The ThalassoChip was updated with new probes and now has the ability to detect 57 ß-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. CONCLUSIONS: The updated version of the ThalassoChip is versatile, robust, cost-effective and easily adaptable, but most notably can provide comprehensive genetic diagnosis for ß-thalassaemia and other haemoglobinopathies.

Hemoglobin variants in Cyprus.

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 beta-globin gene and three concerning the alpha-globin gene. The most common beta-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 alpha-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.

Hb Agrinio [alpha29(B10)Le-->uPro (alpha2)] in combination with --(MED I). Results in a severe form of Hb H disease.

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.