Implementation of multiplex ligation-dependent probe amplification (MLPA) for thalassemia causing deletions has lead to the detection of new rearrangements. Knowledge of the exact breakpoint sequences should give more insight into the molecular mechanisms underlying these rearrangements, and would facilitate the design of gap-PCRs. We have designed a custom fine-tiling array with oligonucleotides covering the complete globin gene clusters. We hybridized 27 DNA samples containing newly identified deletions and nine positive controls. We designed specific primers to amplify relatively short fragments containing the breakpoint sequence and analyzed these by direct sequencing. Results from nine positive controls showed that array comparative genomic hybridization (aCGH) is suitable to detect small and large rearrangements. We were able to locate all breakpoints to a region of approximately 2 kb. We designed breakpoint primers for 22 cases and amplification was successful in 19 cases. For 12 of these, the exact locations of the breakpoints were determined. Seven of these deletions have not been reported before. aCGH is a valuable tool for high-resolution breakpoint characterization. The combination of MLPA and aCGH has lead to relatively cheap and easy to perform PCR assays, which might be of use for laboratories as an alternative for MLPA in populations where only a limited number of specific deletions occur with high frequency.

There is a Blood Commentary on this article in this issue. 8. Brissot E, Labopin M, Beckers MM, et al. Tyrosine kinase inhibitors improve long-term outcome of allogeneic hematopoietic stem cell transplantation for adult patients with Philadelphia chromosome positive acute lymphoblastic leukemia. Haematologica. 2015;100(3):392-399. 9. Zhang FH, Ling YW, Zhai X, et al. The effect of imatinib therapy on the outcome of allogeneic stem cell transplantation in adults with Philadelphia chromosome-positive acute lymphoblastic leukemia. Hematology. 2013; 18(3):151-157. 10. Pfeifer H, Wassmann B, Bethge W, et al; GMALL Study Group. Randomized comparison of prophylactic and minimal residual disease-triggered imatinib after allogeneic stem cell transplantation for BCR-ABL1-positive acute lymphoblastic leukemia. Leukemia. 2013;27(6):1254-1262. 11. Kebriaei P, Saliba R, Rondon G, et al. Long-term follow-up of allogeneic hematopoietic stem cell transplantation for patients with Philadelphia chromosome-positive acute lymphoblastic leukemia: impact of tyrosine kinase inhibitors on treatment outcomes. Biol Blood Marrow Transplant. 2012;18(4):584-592. 12. Short NJ, Jabbour E, Sasaki K, et al. Impact of complete molecular response on survival in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia. Blood. 2016;128(4):504-507. 13. Soverini S, Vitale A, Poerio A, et al. Philadelphia-positive acute lymphoblastic leukemia patients already harbor BCR-ABL kinase domain mutations at low levels at the time of diagnosis. Haematologica. 2011;96(4):552-557. 14. Soverini S, De Benedittis C, Papayannidis C, et al. Drug resistance and BCR-ABL kinase domain mutations in Philadelphia chromosomepositive acute lymphoblastic leukemia from the imatinib to the secondgeneration tyrosine kinase inhibitor era: the main changes are in the type of mutations, but not in the frequency of mutation involvement. Cancer. 2014;120(7):1002-1009.

No abstract

Hemoglobinopathies are the most common monogenic disorders in the world with an ever increasing global disease burden each year. As most hemoglobinopathies show recessive inheritance carriers are usually clinically silent. Programmes for preconception and antenatal carrier screening, with the option of prenatal diagnosis are considered beneficial in many endemic countries. With the development of genetic tools such as Array analysis and Next Generation Sequencing in addition to state of the art screening at the hematologic, biochemic and genetic level, have contributed to the discovery of an increasing number of rare rearrangements and novel factors influencing the disease severity over the recent years. This review summarizes the basic requirements for adequate carrier screening analysis, the importance of genotype-phenotype correlation and how this may lead to the unrevealing exceptional interactions causing a clinically more severe phenotype in otherwise asymptomatic carriers. A special group of patients are β-thalassemia carriers presenting with features of β-thalassemia intermedia of various clinical severity. The disease mechanisms may involve duplicated α-globin genes, mosaic partial Uniparental Isodisomy of chromosome 11p15.4 where the HBB gene is located or haploinsufficiency of a non-linked gene SUPT5H on chromosome 19q, first described in two Dutch families with β-thalassemia trait without variants in the HBB gene.

In Patient 2 no variants or deletions were found that could cause a-thalassemia or bthalassemia, and no large deletions associated with HPFH were present. In the HBG1 promoter a heterozygous 4 bp deletion HBG1:c.253-225_-53-222delAGCA (e.g. 2225_-222delAGCA) was present. This deletion is associated with relatively low expression of the HBG1 gene [6]. Finally, we genotyped SNPs in the HBS1L-MYB and BCL11A loci that are associated with variations in HbF levels in adults [1]. We observed that the SNP genotypes at the HBS1L-MYB and BCL11A loci were not particularly favorable for high HbF expression. In summary, we report hematological parameters of two unrelated patients with microdeletion of 19p13.2-p13.12/13. The commonly deleted region between the patients encompasses 728 kb and contains 33 protein-coding genes including KLF1.In the two patients reported here, the remaining KLF1 allele encodes a wildtype protein.The HbF levels of 7.1% and 16.8% can therefore not be attributed to the presence of a KLF1 variant on the remaining allele. In a Maltese pedigree in which HPFH was caused by KLF1 haploinsufficiency [2], HbF levels varied from 3.3% to 19.5% between HPFH individuals. Thus, the HbF levels of our two patients fall within this range. There is currently no explanation for this high variability in HbF levels. The two patients reported here are the first in which the entire KLF1 gene is deleted. We note that the common deleted region contains four genes (DNASE2, KLF1, LYL1, PRDX2) with known roles in erythropoiesis, and one (NFIX) that was recently linked to fetal hemoglobin levels in a population study (Supporting Information Fig. 1). It remains to be established whether haploinsufficiency for any of these genes contributes to the erythroid phenotypes of the patients. In addition, HBG1/2 promoter variants may contribute to HbF levels in the genes deleted in both patients, DNASE2, LYL1, PRDX2 and NFIX. With the advent of CRISPR/Cas9 genome editing technology for the study of human erythropoiesis, systematic analysis of the roles of these genes in human erythroid cells will be the topic of future research in our laboratories.