Direct sequencing of all fragments was carried out in an automatic sequencer. All sequence variations identified were verified on the complementary strand using an independent PCR product. Multiplex ligand-dependent probe amplification (MLPA) technique for mutations in the RPS19 gene.  Selleck GSI-IX The MLPA technique, which is used for the detection of complete or partial gene deletions or duplications, was carried out [13,14]. This technique is

based on the simultaneous hybridization and ligation of several probes matched to single exons using a single reaction tube, which is followed by PCR and analysis by capillary electrophoresis. Reduced peaks suggest deletions (even on only one exon of a single allele) and enhanced peaks suggest duplication [14]. Informed consent for genetic testing was obtained from the patient and the study was approved by the Trust’s Research and Development Department. Results of genetic analyses.  No loss-of-function mutations were identified in RPS19,

RPS24, RPS17, RPS5, BAY 80-6946 in vivo RPL11 and RPL35a genes that is in keeping with approximately 50% of cases of DBA where no mutations are found in these genes (RPS: ribosomal protein small subunit; RPL: ribosomal protein large subunit). However, heterozygous polymorphisms were identified in RPS24 and RPS17 genes: RPS24 IVSI +26 (c > t); RPS17 IVS2 −73 (g > c), IVS2 −30 (c > t) and nt159 T > C; and homozygous polymorphisms were identified in RPL11 gene: RPL11 −17 (c > g) and IVS5 +39

(a > g) (Fig. 2). The MLPA technique did not reveal any deletion (complete or partial) or duplication in the RPS19 gene (Fig. 3). Implications.  This illustrates a ribosomopathy in a patient with DBA (anaemia, raised adenosine deaminase levels) who subsequently developed CVID. She was dependent on corticosteroids and blood transfusions but went into remission at the age of 6 years. The current definition of ‘remission’ is stable, physiologically acceptable haemoglobin maintained for a minimum of 6 months without corticosteroids, transfusions or other therapy [15]. T cell responses to mitogens were suboptimum, as in a previous case of DBA, which also showed failure of T cell proliferation to human PRKACG recombinant interleukin (rIL)-2 [16]. Our patient therefore resembles approximately half of DBA patients who do not have mutations in the currently described six ribosomal genes (RPS19, RPS17, RPS24, RPL5, RPL11 and RPL35a), but the laboratory abnormalities (anaemia, raised eADA levels) suggest that other genes affecting ribosomal functions may be involved. A recent paper has described mutations in other genes, RPS7, RPS27A, RPL36 and RPS15, evident in DBA, but we have not looked for mutations in these genes [8].