Characterization of 17 new cases of X-linked chronic granulomatous disease with seven novel mutations in the CYBB gene

Objective: Molecular identification and clinical characterization of genetic mutations in patients with X-linked chronic granulomatous disease (CGD). Patients and methods: Genomic DNA from 17 male patients with proven X-linked CGD based on clinical history, clinical examination, and specific granulocyte function tests were amplified by polymerase chain reaction (PCR) for sequences of the CYBB gene encoding gp91-phox. Mutations in the resulting PCR products were identified by DNA sequencing. Results: Sequence analysis revealed four deletions (453_454delAG; 802delC; 962delG; 993delG), one combined deletion/insertion/duplication [156_173delTCAGCACTGGCACTGGCC/174_175insT/175_176insCCTGCCTGAATTTCT(dupl187_200)]; one insertion (574_575insCCTCAT), four nonsense mutations (332G > A; 402C > T; 690C > T; 1340C>G), two missense mutations (933A > C; 1041A > C) and four potential splice-site mutations (5'intron1 gt-->at; 3'intron1ag-->at; 5'intron3 gtaag-->gtaaa; 5'intron4 gtaa-->ctaa). Seven of these mutations were indeed novel, whereas four mutations not previously reported to the X-CGDbase were found to be of the same type as database reports of unrelated families. The six remaining mutations have been reported previously to the X-CGDbase but have as yet not been described in detail. Conclusion: Our findings underline the great heterogeneity of mutations involving the CYBB gene. Neither a mutational hot spot in the gp91-phox gene nor a clear correlation between molecular defect and clinical manifestation in unrelated families could be demonstrated. Remarkable is a splice-site mutation (5'intron3 gtaag-->gtaaa) identified in a 40-year-old patient with late onset "adult" CGD and residual nicotinamide adenine dinucleotide phosphate reduced oxidase activity. The enormous delay of clinical symptoms of this particular mutation could be explained by an age-related variable sensitivity of the splicing machinery to the present splice-site mutation

Microbubbles in macrocysts – Contrast-enhanced ultrasound assisted sclerosant therapy of a congenital macrocystic lymphangioma: a case report

Abstract Background Congenital cystic lymphangiomas are benign malformations due to a developmental disorder of lymphatic vessels. Besides surgical excision, sclerosant therapy of these lesions by intracavitary injection of OK-432 (Picibanil®), a lyophilized mixture of group A Streptococcus pyogenes, is a common therapeutical option. For an appropriate application of OK-432, a detailed knowledge about the structure and composition of the congenital cystic lymphangioma is essential. SonoVue® is a commercially available contrast agent commonly used in sonography by intravenous and intracavitary application. Case presentation Here we report the case of 2 month old male patient with a large thoracic congenital cystic lymphangioma. Preinterventional imaging of the malformation was performed by contrast-enhanced ultrasound after intracavitary application of SonoVue® immediately followed by a successful sclerotherapy with OK-432. Conclusions Contrast agent-enhanced ultrasound imaging offers a valuable option to preinterventionally clarify the anatomic specifications of a congenital cystic lymphangioma in more detail than by single conventional sonography. By the exact knowledge about the composition and especially about the intercystic communications of the lymphangioma sclerosant therapy becomes safer and more efficient

Cluster analysis of genomic ETV6-RUNX1 (TEL-AML1) fusion sites in childhood acute lymphoblastic leukemia

Fusion between ETV6 and RUNX1 defines the largest genetic subgroup in childhood ALL The genomic fusion site, unique to individual patients and specific for the malignant clone, represents an ideal molecular marker for quantification of minimal residual disease. Sequencing of DNA breakpoints has been difficult due to the extended size of the respective breakpoint cluster regions. We therefore evaluated a specially designed multiplex long-range PCR assay in 65 diagnostic bone marrow samples for its suitability in routine use. Resulting fusion sites and breakpoints derived from previous studies were subject to cluster analysis to identify potential sequence motifs involved in translocation initiation. (C) 2008 Elsevier Ltd. All rights reserved

HLA-B27-restricted T cells from patients with ankylosing spondylitis recognize peptides from B*2705 that are similar to bacteria-derived peptides

Ankylosing spondylitis (AS) is an inflammatory systemic disease affecting the spine, sacroiliacal and peripheral joints. Although the aetiology of AS remains unknown, the strong association with the HLA-B27 allele might reflect directly a detrimental effect of the HLA-B27 molecule itself, resulting from its potential capability to present ‘arthritogenic’ peptides to CD8(+) T cells. Because some forms of SpA are triggered by enterobacterial infection, such arthritogenic peptides might originate from autologous and/or bacterial proteins triggering cross-reactive CD8(+) T cell clones. Intriguingly, two peptides from the second extracellular domain of HLA-B*2705 share sequence homologies with several enterobacterial antigens, exhibit the HLA-B27-binding-motif, and are presented by HLA-B*2705 itself. The objective of this study was to examine the clonal T cell reactivity against these peptides in patients with AS. To this end, we screened peripheral blood lymphocytes (PBL) of 26 patients with AS and 24 healthy donors for TNF-α-producing cells using ELISPOT assays. PBL and synovial fluid-derived lymphocytes (SFL) of peptide-responsive patients were then stimulated and cultured with the relevant peptide and control peptides in vitro. Antigen-specific T cell lines (TCL) were identified by standard chromium release assays. Clonal analysis was performed subsequently applying TCRB-CDR3 spectratyping. Among eight peptides tested, only the HLA-B27 168–176 peptide LRRYLENGK was recognized by PBL from B27(+) AS patients but not from B27(+) healthy controls (P = 0·001). LRRYLENGK-specific T cell clones used preferentially the TCRBV5S1 and the BV14 segment. These results suggest that an HLA-B27-derived peptide with homology to bacterial peptides may play a role in AS