Mutations in the TET2 gene are frequent in myeloid disease, although their biologic and prognostic significance remains unclear. We analyzed 355 patients with myelodysplastic syndromes using "next-generation" sequencing for TET2 aberrations, 91 of whom were also subjected to single-nucleotide polymorphism 6.0 array karyotyping. Seventy-one TET2 mutations, with a relative mutation abundance (RMA) > 10%, were identified in 39 of 320 (12%) myelodysplastic syndrome and 16 of 35 (46%) chroni myelomonocytic leukemia patients (P < .001).Interestingly, 4 patients had multiple mutations likely to exist as independent clones or on alternate alleles, suggestive of clonal evolution. "Deeper" sequencing of 96 patient samples identified 4 additional mutations (RMA, 3%-6.3%). Importantly, TET2 mutant clones were also found in T cells, in addition to CD34 ؉ and total bone marrow cells (23.5%, 38.5%, and 43% RMA, respectively). Only 20% of the TET2-mutated patients showed loss of heterozygosity at the TET2 locus. There was no difference in the frequency of genome-wide aberrations, TET2 expression, or the JAK2V617F 46/1 haplotype between TET2-mutated and nonmutated patients. There was no significant prognostic association between TET2 mutations and World Health Organization subtypes, International Prognostic Scoring System score, cytogenetic status, or transformation to acute myeloid leukemia. On multivariate analysis, age (> 50 years) was associated with a higher incidence of TET2 mutation (P ؍ .02).
Beta thalassaemia intermedia due to co-inheritance of three unique alpha globin cluster duplications characterised by next generation sequencing analysis Co-inheritance of a thalassaemia reduces chain imbalance and disease severity in b thalassaemia homozygotes, while increasing a globin output in heterozygotes increases chain imbalance, converting a typically asymptomatic carrier state to that of thalassaemia intermedia. The outcome depends on the number of a globin genes inherited as one or two copies of triplicated (/aaa), or quadruplicated (/aaaa) a globin complexes, and the type of b thalassaemia mutation (b 0 or b + ) (Thein, 2008). Another mechanism of increasing a globin output is through segmental duplication of the whole a globin complex (Harteveld et al, 2008) but breakpoints of the reported duplications have not been fully characterised due to technological limitations. Here, we applied a previously described next generation sequencing (NGS) methodology (Shooter et al, 2015a,b) to characterise three a globin cluster duplications, permitting to-the-base resolution in two of the three cases. Patient samples were referred for work-up of unusually severe phenotype in b thalassaemia carriers. In Family 1 (Fig 1A), the proband was a 54-year-old Chinese male with hypochromic microcytic anaemia since infancy. His partner (Anglo-Saxon English) and two older sons had normal haematological profiles; the youngest son (aged 14 years) had a haematological profile similar to that of the father. Both father and son had (Fig 1C) is due to iron deficiency.Correspondence 160 ª
Genetic testing for hemoglobinopathies is required for prenatal diagnosis, understanding complex cases where multiple pathogenic variants may be present or investigating cases of unexplained anemia. Characterization of disease causing variants that range from single base changes to large rearrangements may require several different labor-intensive methodologies. Multiplex ligation probe amplification analysis is the current method used to detect indels, but the technique does not characterize the breakpoints or detect balanced translocations. Here, we describe a next-generation sequencing (NGS) method that is able to identify and characterize a novel rearrangement of the HBB cluster responsible for εγδβ thalassemia in an English family. The structural variant involved a 59.0 kb inversion encompassing HBG2 exon 3, HBG1, HBD, HBB, and OR51V1, juxtaposed by a deletion of 122.6 kb including 82 bp of the inverted sequence, HBG2 exon 1 and 2, HBE, and the β-locus control region. Identification of reads spanning the breakpoints provided to-the-base resolution of the rearrangement, subsequently confirmed by gap-PCR and Sanger sequence analysis. The same rearrangement, termed Inv-Del English V εγδβ thalassemia (HbVar 2935), was identified in two other unrelated English individuals with a similar hematological phenotype. Our NGS approach should be applicable as a diagnostic tool for other disorders.
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