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
lymphocytosis to those without del(17p), suggesting that the effect of T12 on lymphocytosis pattern is dominant; this is consistent with the prior observation that up-regulation of integrin signalling in T12 is not modulated by additional del (11q) or del(17p) (Riches et al, 2014). We did not have confirmatory integrin expression data. In contrast to the effect on lymphocytosis pattern, the presence of T12 in addition to del (17p) did not attenuate the adverse impact of del(17p) on outcomes. Further studies are required to ascertain the mechanisms underlying the abbreviated lymphocytosis in T12 CLL and its relationship, if any, to therapeutic efficacy. AcknowledgementsThe authors would like to acknowledge Ms. Susan Smith for assistance in data gathering and management. AuthorshipPT provided clinical care to patients, collected and analysed data, performed statistical analysis and wrote the paper. AF, SOB, WGW, MJK and JAB provided clinical care to patients, assisted in the analysis of data and development of critical themes and coauthored the paper. A 26-year-old West African woman underwent routine antenatal screening for haemoglobinopathies. Her blood counts showed Hb 119 g/l, RBC 4Á45 9 10 9 /l, MCV 77Á2 fl, MCH 26Á8 pg. High performance liquid chromatography (BioRad variant II, Hercules, CA, USA) screen identified a haemoglobin variant in the HbS position comprising 13Á5%, HbF 0Á2% plus normal adult haemoglobins. When separated at acid pH the unknown variant migrated to the HbS position but this could not be confirmed by a solubility assay due to the low HbS percentage. Conflict of interestDNA was extracted from EDTA whole blood using a Qiagen Symphony midi kit (Qiagen, Hilden, Germany) and from a saliva sample (Oragene kit, Genotek, Ontario, Canada) using a Qiagen Virus kit (v2) on an EZ1 Biorobot (Qiagen). Genomic DNA was analysed for variants in the HBA and HBB genes, as described (Clark & Thein, 2004). Genetic testing showed that the individual had the aa/aÀ 3Á7 genotype and no alpha thalassaemia variants. Sequence analysis of the HBB genes did not identify any b thalassaemia mutations that could have been in cis with the HBB p.Glu7Val mutation, which would have explained the unusually low HbS percentage. We confirmed the HBB p.Glu7Val mutation by TaqMan analysis but the relative proportions of the sickle and normal alleles in the gene sequencing and TaqMan assay suggested the presence of an additional functioning normal HBB allele. A next generation sequencing (NGS) assay and bioinformatic strategy was used to identify any potential rearrangement in the HBA and HBB globin gene loci that could explain the unusually low HbS (Shooter et al, 2014). Leucocyte-extracted DNA (3 lg) was sheared to 500 bp using a Bioruptor (Diagenode, Denville, NJ, USA) and used to construct a sequencing library with minimal polymerase chain reaction (PCR) amplification. In-solution bait capture (Agilent, Santa Clara, CA, USA) target enrichment isolated DNA fragments from two genomic regions, 4 Mb on chromosome 11 encompassing...
The gene for hereditary persistence of fetal hemoglobin (HPFH) in the Caribbean is much more common than previously estimated. To avoid labeling persons with the benign syndrome Hb S (HBB: c.20A>T)/HPFH as a disease and wasting scarce resources, parental studies are recommended when newborn screening reveals a pattern consistent with an SS phenotype.
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