Identification of Heterozygous Single- and Multi-exon Deletions in IL7R by Whole Exome Sequencing

PURPOSE: We aimed to achieve a retrospective molecular diagnosis by applying state-of-the-art genomic sequencing methods to past patients with T-B+NK+ severe combined immunodeficiency (SCID). We included identification of copy number variations (CNVs) by whole exome sequencing (WES) using the CNV calling method ExomeDepth to detect gene alterations for which routine Sanger sequencing analysis is not suitable, such as large heterozygous deletions. METHODS: Of a total of 12 undiagnosed patients with T-B+NK+ SCID, we analyzed eight probands by WES, using GATK to detect single nucleotide variants (SNVs) and small insertions and deletions (INDELs) and ExomeDepth to detect CNVs. RESULTS: We found heterozygous single- or multi-exon deletions in IL7R, a known disease gene for autosomal recessive T-B+NK+ SCID, in four families (seven patients). In three families (five patients), these deletions coexisted with a heterozygous splice site or nonsense mutation elsewhere in the same gene, consistent with compound heterozygosity. In our cohort, about a quarter of T-B+NK+ SCID patients (26%) had such compound heterozygous IL7R deletions. CONCLUSIONS: We show that heterozygous IL7R exon deletions are common in T-B+NK+ SCID and are detectable by WES. They should be considered if Sanger sequencing fails to detect homozygous or compound heterozygous IL7R SNVs or INDELs

Treosulfan, Fludarabine Conditioning for HSCT in Children with Primary Immunodeficiency: UK Experience

We previously published results of 70 children who received treosulfan with cyclophosphamide (30) or fludarabine (40) before haematopoietic stem cell transplantation (HSCT) for Primary Immunodeficiency (PID). Toxicity was lower and T cell chimerism better in those receiving fludarabine, but numbers were relatively small and follow-up short. We now report outcome of 160 children who received homogeneous conditioning with treosulfan, fludarabine mostly with alemtuzumab (n=124). Median age at transplant was 1.36 years (0.09-18.25). Donors were: matched unrelated, 73; 1-3 antigen mismatched unrelated, 54; matched sibling, 12; other matched family, 17; haploidentical, 4. Stem cell source was: peripheral blood stem cells (PBSCs), 70; Bone marrow, 49; Cord Blood, 41. Median follow up was 4.3 years (0.8-9.4). Overall survival was 83%. There was no veno- occlusive disease. Seventy-four (46%) had acute GVHD, but only 14(9%) greater than grade II. Four patients were successfully retransplanted for graft loss or poor immune reconstitution. One further patient who rejected the graft, died. There was no association between T cell chimerism > 95% and stem cell source, but a significant association with myeloid chimerism > 95% and use of PBSC without an increased risk of significant GVHD compared to other sources. All 11 patients with severe combined immunodeficiency diagnosed at birth are alive with up to 8.7 years follow up. Long-term studies are required to determine late gonadotoxic effects and pharmacokinetic studies are needed to identify whether specific targeting is advantageous. The combination of treosulfan, fludarabine and alemtuzumab gives excellent results in HSCT for PID

The Potential Role of ORM2 in the Development of Colorectal Cancer

Colorectal cancer (CRC) is the third most common malignancy in the world. The risk of death is closely correlated to the stage of CRC at the time of primary diagnosis. Therefore, there is a compelling need for the identification of blood biomarkers that can enable early detection of CRC. We used a quantitative proteomic approach with isobaric labeling (iTRAQ) to examine changes in the plasma proteome of 10 patients with CRC compared to healthy volunteers. Enzyme-Linked Immunosorbnent Assay (ELISA) and Western blot were used for further validation. In our quantitative proteomics analysis, we detected 75 human plasma proteins with more than 95% confidence using iTRAQ labeling in conjunction with microQ-TOF MS. 9 up-regulated and 4 down-regulated proteins were observed in the CRC group. The ORM2 level in plasma was confirmed to be significantly elevated in patients suffering from CRC compared with the controls. ORM2 expression in CRC tissues was significantly increased compared with that in corresponding adjacent normal mucous tissues (P<0.001). ITRAQ together with Q-TOF/MS is a sensitive and reproducible technique of quantitative proteomics. Alteration in expression of ORM2 suggests that ORM2 could be used as a potential biomarker in the diagnosis of CRC