Genetic studies of three Japanese patients with p22-phox-deficient chronic granulomatous disease: detection of a possible common mutant CYBA allele in Japan and a genotype-phenotype correlation in these patients.

Chronic granulomatous disease (CGD) is a disorder caused by defects in the NADPH oxidase responsible for superoxide generation in phagocytes. Cytochrome b558, an essential component of this enzyme, is a heterodimer formed by a 91 kDa glycoprotein (gp91-phox) and a 22 kDa polypeptide (p22-phox). Mutations in the p22-phox gene (CYBA) locus in 16q24 result in one of the rare autosomal recessive forms of CGD. We performed mutation analysis in three female CGD patients suspected of having this form of the disease and found two novel mutations in CYBA. Whereas patient 1 with severe phenotype had a homozygous nonsense mutation in exon 1 (C-35 --> T, Gln-3 --> stop), patients 2 and 3 with mild phenotype shared the same homozygous missense mutation in exon 2 (G-98 --> A, Gly-24 --> Arg). None of the parents of patients 2 and 3 is related. Therefore, this mutation could be a hot-spot or a common mutation in the Japanese population. Patients 2 and 3, but not patient 1, were demonstrated to have detectable p22-phox expression and significant granulocyte respiratory burst (ROB) activity. In this study, we were able to demonstrate an excellent correlation between genotype, p22-phox expression, ROB activity and clinical phenotype in these patients.

Identification of a genetic risk factor for systemic juvenile rheumatoid arthritis in the 5'-flanking region of the TNFalpha gene and HLA genes.

OBJECTIVE: To study polymorphisms in the 5'-flanking promoter/enhancer region of the tumor necrosis factor alpha (TNFalpha) gene and in the coding regions of HLA class I and class II genes, in order to better understand the genetic background of juvenile rheumatoid arthritis (JRA). METHODS: One hundred eleven Japanese JRA patients (50 with systemic disease, 29 with pauciarticular disease, and 32 with polyarticular disease) and 575 healthy Japanese subjects were examined for the allele frequencies of the TNFalpha, HLA-A, and HLA class II (DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPA1, and DPB1) genes, by DNA typing using the polymerase chain reaction-sequence-specific oligonucleotide probe method. RESULTS: The frequencies of the polymorphic allele at positions -1,031 (T to C substitution, termed -1,031C), -863 (C to A, termed -863A), and -857 (C to T, termed -857T) of the TNFalpha gene in patients with systemic JRA, but not in those with polyarticular or pauciarticular JRA, were significantly higher than in the healthy controls. The allele frequencies of DRB1*0405 and DQB1*0401 in systemic JRA, but not in the other JRA types, were significantly higher than in controls. Linkage analysis showed that the presence of both the TNFalpha -857T allele and DRB1*0405 yielded a significantly increased odds ratio (3.84), while the presence of only 1 of them did not yield a high odds ratio (0.87 and 1.58). CONCLUSION: The -1,031C/-863A allele and the -857T allele of the TNFalpha gene, both of which are related to high production of tumor necrosis factor alpha, are associated with systemic JRA. The -857T allele may enhance the effect of the DRB1*0405/DQB1*0401 haplotype in predisposing to development of systemic JRA.

Mutation at histidine 338 of gp91(phox) depletes FAD and affects expression of cytochrome b558 of the human NADPH oxidase.

Defective NADPH oxidase components prevent superoxide (O-2) generation, causing chronic granulomatous disease (CGD). X-linked CGD patients have mutations in the gene encoding the gp91(phox) subunit of cytochrome b558 and usually lack gp91(phox) protein completely (X91(0)). gp91(phox) is considered to be a flavocytochrome that contains binding sites for NADPH, FAD, as well as heme. We here report a rare X-linked CGD patient whose neutrophils entirely failed to produce O-2, but presented a diminished expression of gp91(phox) containing about one-third of the heme present in normal individuals by Soret absorption. Translocation of cytosolic factors p67(phox) and p47(phox) was normal. However, the FAD content in his neutrophil membranes was as low as that of X91(0) patients, suggesting complete depletion of FAD in his gp91(phox). This was in agreement with the finding that a single base substitution (C1024 to T) changed His-338 to Tyr in gp91(phox) in a predicted FAD-binding domain of the flavocytochrome model. The loss of FAD could not be corrected even after addition of reagent FAD or a FAD-rich dehydrogenase fraction isolated from normal neutrophils to the patient's membranes, in a reconstitution in vitro with normal cytosol. These results indicate that His-338 is a very critical residue for FAD incorporation into the NADPH oxidase system. This is the first such mutation found in CGD.

[Twelve cases of systemic lupus erythematosus in boys].

We compared clinical findings in 12 cases of systemic lupus erythematosus (SLE) in boys with those in 49 cases in girls. The age at which SLE developed in boys was consistent with that of infantile SLE and there was no age specificity. Momy cases in boys were diagnosed earlier as compared with cases in girls. Symptoms of infantile SLE, such as fever, arthalgia, butterfly rash, and urinary abnormalities, did not differ between boys and girls. However, a higher percentage of boys (58.3%) had central nervous system complications at onset than did girls (30.6%). Platelet counts tended to be higher in boys than in girls, a finding that suggests SLE tends to be more severe in boys than in girls. The incidence in the appiarance of LE cells, anti-Sm antibodies and immune complexes was higher in boys than in girls. Type IV or V renal pathologic changes (World Health Organization Histologic Classification) were present in 70% of boys. Our findings suggest that SLE in boys is more severe than that in girls and is more likely to be associated with central nervous system complications and severe renal complications.

Deficient expression of Bruton's tyrosine kinase in monocytes from X-linked agammaglobulinemia as evaluated by a flow cytometric analysis and its clinical application to carrier detection.

The B-cell defect in X-linked agammaglobulinemia (XLA) is caused by mutations in the gene for Bruton's tyrosine kinase (BTK). Using the anti-BTK monoclonal antibody (48-2H), a flow cytometric analysis of intracytoplasmic BTK protein expressed in monocytes was successfully performed. To examine the possible identification of XLA patients and female carriers by this assay, we studied 41 unrelated XLA families with (35) or without (6) known BTK mutations. A flow cytometric assay showed deficient expression of the BTK protein in 40 of 41 patients, complete BTK deficiency in 35, and partial BTK deficiency in 5. One patient exhibited a normal level of BTK expression. All 6 patients with partial BTK deficiency or normal BTK expression had missense BTK mutations. The cellular mosaicism of BTK expression in monocytes from obligate carriers was clearly shown in 35 of 41 families. The results suggested that most BTK mutations in XLA might result in deficient expression of the BTK protein. We conclude that deficient expression of BTK protein can be evaluated by a flow cytometric assay, and the clinical usefulness and limitations in diagnosis of XLA patients and carriers are discussed.

Characteristics of juvenile dermatomyositis in Japan.

Questionnaires were sent to 1290 hospitals in Japan asking for data on patients with juvenile dermatomyositis (JDM) diagnosed between June 1984 and May 1994. Of the 204 patients identified by these questionnaires, 102 met the criteria for JDM. JDM is categorized into three subtypes: Banker-type JDM, Brunsting-type and fulminant-type; patients with the latter exhibit markedly elevated serum levels of creatinine phosphokinase (> 10,000 U/mL) and appear to be at risk of renal failure. Cutaneous manifestations were present in 98% of patients and preceded the appearance of other symptoms. This tendency is one of the reasons for the difficulty in some cases in diagnosing the onset of JDM. Better criteria for early treatment of JDM are needed. The results of the present study suggest that itching and calcinosis are factors that indicate a poor prognosis in patients with JDM. Muscle enzyme levels do not always reflect disease activity, suggesting that methods other than measurement of muscle enzymes, such as measurement of the levels of neoprerin and von Willebrand factor antigen, as well as magnetic resonance imaging should be used to be evaluate disease severity. Patients with Brunsting-type JDM who exhibit dysphagia and antinuclear antibody positivity and patients with Banker-type JDM should be treated aggressively. Pulse therapy should be selected as the initial therapy in patients with fulminant-type JDM.

Two-exon skipping due to a point mutation in p67-phox--deficient chronic granulomatous disease.

The cytosolic 67-kD protein in phagocytes (p67-phox) and B lymphocytes is one of essential components of the superoxide-generating system in these cells, and its defect causes an autosomal recessive type of chronic granulomatous disease (CGD). We performed mutation analysis of p67-phox mRNA from a CGD patient who lacks the protein and found an in-frame deletion from nucleotide 694 to 879, which corresponds to the entire sequence of exons 8 and 9. This sequence encodes one of two Src homology 3 domains and a part of proline-rich domain in p67-phox and lack of these domains seem to have influenced stability of this protein. To know causative reason for the deletion, we analyzed genomic DNA for p67-phox using two sets of primers that covered exons 8 and 9 with adjacent introns. The DNA fragments from the patient were shown to be same in length as those from control. However, the single-strand conformation-polymorphism analysis of the fragments showed that a patient's specimen that included the splice junction of exon 9 exhibited different mobility from the control. By sequencing of the fragment, a homozygous G to A replacement at position +1 of intron 9 was found to be a sole mutation, which reduced the matching score of the splicing sequence to the consensus calculated according to the formula proposed by Shapiro and Senapathy (Nucleic Acids Res 15:7155, 1987). The reduced matching score at the splice doner site (5' splice site) of intron 9 and the original low matching score at the acceptor site (3' splice site) of intron 7 may explain the skipping of exon 8 and 9, and another predicted mechanism is discussed on the basis of Shapiro and Senapathy's hypothesis.

A novel cytosolic component, p40phox, of respiratory burst oxidase associates with p67phox and is absent in patients with chronic granulomatous disease who lack p67phox.

Cytosolic components of human neutrophils, p47phox and p67phox, deficiencies of which lead to chronic granulomatous disease (CGD), potentiate respiratory burst oxidase translocating from cytosol to membrane upon cell stimulation. In this report we describe a novel cytosolic component, p40phox, which consistently behaves with p67phox through immunoprecipitation and column works, and is missing in patients with CGD who lack p67phox. Although actin has been reported to be involved in O2- generation, the p40phox profile did not correspond to that of actin. The tight association between p40phox and p67phox was not affected by treatment with a mixture of deoxycholate and Nonidet P-40, until subjected to SDS-PAGE. Addition of recombinant p67phox to cytosol did not produce any additional p40phox in the immunoprecipitate, unlike the additive increment in the band of p67phox. These results suggest that p40phox forms a complex with p67phox in a molar ratio of 1:1, without any free p40phox in the cytosol.

[Clinical study of systemic juvenile rheumatoid arthritis (Still)].

Thirty-seven patients with systemic JRA were analyzed. Fifty four per cent of patients had mono-cyclic systemic type. Age at onset ranged from 6.0-6.8 years (median 6.4). Boys were more affected than girls (24/11). Cardiac involvement occurred in 10 patients (27%). Patients with cardiac troubles showed significantly much number of the white blood cell counts at admission and the max white blood cell count than those without cardiac troubles. Duration of positive CRP was shorter in patients with cardiac involvement who were all given cortico-steroid hormone those without cardiac involvement. This means that it is better to use steroid hormone early for patients with cardiac involvement. Patients with chronic arthritis type had higher elevated erythrocyte sedimentation rate and serum C3 level at admission and longer duration of positive CRP. We speculated that these date showed inflammation of joints. The onset subtype, which was determined by manifestations during the first 6 months of disease, was important for predicting clinical course and outcome.

[Therapeutic evaluation of combination therapy using C-425, human native immunoglobulin liquid preparation for i.v. administration, and antibiotics in severe and/or refractory infections in pediatrics].

A newly developed human immunoglobulin liquid preparation for intravenous injection was studied for efficacy, safety, and usefulness in treating severe and/or refractory infections in children receiving antibiotic treatment. It is suggested that C-425 is a useful intravenous preparation of human immunoglobulin for the treatment of severe and/or refractory infections in pediatrics. C-425 was administered to 87 inpatients with severe and/or refractory infections at 23 institutions nationwide. The Committee selected 61 cases for the present analysis. Physicians in charge judged clinical efficacy of C-425 to be "excellent" in 23 cases (40.4%), "good" in 24 (42.1%), "fair" in 7 (12.3%), "poor" in 3 (5.3%), and "unknown" in 4. The efficacy rate was calculated at 82.5% when the "excellent" and "good" cases were combined, and 94.7% when the "fair" cases were also included. According to the Committee's judgement, the efficacy of C-425 was "excellent" in 27 cases (44.3%), "good" in 18 (29.5%), "fair" in 7 (11.5%), and "poor" in 9 (14.8%). The efficacy rate was 73.8% when the "excellent" and "good" cases were combined. The rate increased to 85.2% when the "fair" cases were added. Organisms were identified in 31 cases, and the time course was followed in 19 instances. Organisms were eliminated in 12 cases (63.2%), decreased in number in 2 (10.5%), and persisted in 5 (26.3%). Eradication rate was 63.2%. One of the 87 patients died of fulminant hepatitis 2 days after the end of the treatment. The remaining 86 cases were analyzed for the safety of C-425. A skin rash was observed in one case. Laboratory examination revealed increase in transaminase levels in a total of 8 cases; both in GOT and GPT in 5, in GOT alone in 2, and in GPT alone in 1. These findings were not clinically important.

[Clinical and fundamental studies on intravenous drip infusion of gentamicin in the pediatric field. Pediatric study group of gentamicin].

A multiclinic study of gentamicin (GM) given by intravenous drip infusion was carried out by the Gentamicin Pediatric Study Group. The results are summarized as follows: 1. Upon intravenous drip infusion of GM at a dose range of 2.0-2.5 mg/kg over a period of 0.5-1 hour, therapeutically effective serum concentrations of 4-12 micrograms/ml were obtained. These values are similar to reported values in previous studies using GM intramuscular injection. 2. High urinary concentrations were observed up to 6 hours after administration, and the urinary recovery rate was approximately 60%. 3. Of a total of 142 cases collected, 117 cases were evaluated. Efficacy rates by diseases were: 100% in pneumonia (30/30), 98.3% in urinary tract infections (59/60), and 92.3% in other infections (skin and soft tissue) (12/13), with an overall efficacy rate of 94.9% (including 77 "excellent" cases). 4. Bacteriological examinations showed high eradication rates with the use of GM; i.e., 80% with Staphylococcus aureus (8/10), 60% with Pseudomonas aeruginosa (3/5), 100% with Haemophilus influenzae (7/7) and 97.8% with Escherichia coli (44/45), achieving an overall eradication rate of 92.4%. In mixed infections, the eradication rate was 85.7% (6/7). 5. No ototoxicity, nephrotoxicity or allergic reactions was observed. Abnormal laboratory findings observed were: GOT elevation in 3.1% of cases, GPT elevation in 3.9%, platelet increase in 1.5% and eosinophil increase in 0.8%, thus an overall rate of the appearance of abnormality was 5.6%. The above results indicate that an intravenous drip infusion of GM is a useful method for treating infections in pediatrics.

Detection of antibodies to human parvovirus in erythema infectiosum (fifth disease).

Two Japanese outbreaks of erythema infectiosum were investigated for evidence of human parvovirus infection by a solid phase antibody capture radioimmunoassay based on a monoclonal antibody to human parvovirus. Specific IgM and high concentrations of specific IgG were detected in 37 sera from 27 children with erythema infectiosum. No anti human parvovirus IgM was detected in a remaining case of erythema infectiosum, in five patients with Kawasaki disease, or in the 17 control children. Seven of the controls were also anti human parvovirus IgG negative, and the 10 who were seropositive had lower concentrations of anti human parvovirus IgG than the patients with erythema infectiosum. These data indicate that human parvovirus is a cause of erythema infectiosum.