Characterization of six novel mutations in the CYBB gene leading to different sub-types of X-linked chronic granulomatous disease.

Chronic granulomatous disease is an inherited disorder in which phagocytes lack a functional NADPH oxidase and so cannot generate superoxide anions (O(2) (-)). The most common form is caused by mutations in CYBB encoding gp91 phox, the heavy chain of flavocytochrome b(558) (XCGD). We investigated 11 male patients and their families suspected of suffering from X-linked CGD. These XCGD patients were classified as having different variants (X91(0), X91(-) or X91(+)) according to their cytochrome b(558) expression and NADPH oxidase activity. Nine patients had X91(0) CGD, one had X91(-) CGD and one had X91(+) CGD. Six mutations in CYBB were novel. Of the four new X91(0) CGD cases, three were point mutations: G65A in exon 2, G387T in exon 5 and G970T in exon 9, leading to premature stop codons at positions Try18, Try125 and Glu320, respectively, in gp91 phox. One case of X91(0) CGD originated from a new 1005G deletion detected in exon 9. Surprisingly, four nonsense mutations in exon 5 led to the generation of two mRNAs, one with a normal size containing the mutation and the other in which exon 5 had been spliced. A novel X91(-) CGD case with low gp91 phox expression was diagnosed. It was caused by an 11-bp deletion in the linking region between exon 12 and intron 12, activating a new cryptic site. Finally, a new X91(+) CGD case was detected, characterized by a missense mutation Leu505Arg in the potential NADPH-binding site of gp91 phox. No clear correlation between the severity of the clinical symptoms and the sub-type of XCGD could be established.

Urinary release of 72 and 92 kDa gelatinases, TIMPs, N-GAL and conventional prognostic factors in urothelial carcinomas.

OBJECTIVES: A urinary release of gelatinases A and B matrix metalloproteinases-2, -9 (MMP-2, -9), and tissue inhibitors (TIMP-1, -2) occurs during normal epithelial turnover. A proteinase increase, reduced inhibitors or both potentially account for cell mobility and bladder cancer progression. In order to define normal levels and thresholds for transitional cell carcinoma (TCC) patients, urinary gelatinases, tissue inhibitors and neutrophil-gelatinase-associated lipocalin (N-GAL) were investigated for end-point clinical status and compared with normal subjects during a 2-year follow-up prospective study. METHODS: Urine specimens [50 adult normal controls; 28 in situ carcinoma patients (pTa) and 23 with ruptured basement membrane (pT1-4)] were screened by gelatin zymograms, immunoblots and ELISA. RESULTS: (1) An important release of inhibitors over low levels of active enzymes was observed in controls independently of age and sex except for higher TIMP-1 levels in males. (2) In cancer patients, increased pro-MMP-9 and active MMP-2 with reduced TIMP-2 levels correlated with higher stages and histological grades. (3) Conversely, reduced MMP-9 and lipocalin levels were initial hallmarks of clinical relapses. CONCLUSIONS: The imbalance between increased MMP-2, -9 and decreased TIMP-2 levels appears to be linked to tumor stage and grade and, more importantly, to clinical events. Changes in the MMP-9 activation state and a lack of N-GAL present as novel markers of tumor progression.