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...
Pyruvate kinase (PK) deficiency is an autosomal recessive disease caused by mutations in the PKLR gene, which reduce erythrocyte PK enzyme activity and result in decreased energy synthesis in red cells, causing haemolytic anaemia. Historically, the investigation into pyruvate kinase deficiency (PKD) has been led by a red cell enzyme assay determining PK enzyme activity per unit of haemoglobin. For our laboratory, the reference range was set by Beutler et al. in 1977 when the test was first established. The introduction of genetic testing permitted the creation of reference sample datasets, with positive controls having two pathogenic variants causing disease. This permitted reassessment of the enzyme assay's sensitivity and specificity, and was used to reassess the reference range of the enzyme assay. Using sequenced samples, we have devised an enzyme assay, DNA testing workflow, which minimises false negative/positive results and improves the diagnostic efficiency. This combined enzyme-DNA testing strategy should improve the diagnostic accuracy whilst limiting the number of expensive DNA tests. During this evaluation, 10 novel genetic variants were identified and are described.
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