Loss of Major DNase I Hypersensitive Sites in Duplicated ß-globin Gene Cluster Incompletely Silences HBB Gene Expression.

We report an infant with sickle cell disease phenotype by biochemical analysis whose ß-globin gene (HBB) sequencing showed sickle cell mutation (HBBS ) heterozygosity. The proband has a unique head-to-tail duplication of the ß-globin gene cluster having wild-type (HBBA ) and HBBS alleles inherited from her father; constituting her HBBS /HBBS -HBBA genotype. Further analyses revealed that proband's duplicated ß-globin gene cluster (∼650 kb) encompassing HBBA does not include the immediate upstream locus control region (LCR) or 3' DNase I hypersensitivity (HS) element. The LCR interacts with ß-globin gene cluster involving long range DNA interactions mediated by various transcription factors to drive the regulation of globin genes expression. However, a low level of HBBA transcript was clearly detected by digital PCR. In this patient, the observed transcription from the duplicated, distally displaced HBBA cluster demonstrates that the loss of LCR and flanking 3'HS sites do not lead to complete silencing of HBB transcription.

Next generation sequencing identifies a novel rearrangement in the HBB cluster permitting to-the-base characterization.

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 ÎµÎ³Î´ß 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 ß-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 ÎµÎ³Î´ß 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.

Next-generation sequencing of the TET2 gene in 355 MDS and CMML patients reveals low-abundance mutant clones with early origins, but indicates no definite prognostic value.

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%) chronic 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).