Child, Preschool ; Flow Cytometry ; methods ; Genetic Carrier Screening ; Granulomatous Disease, Chronic ; diagnosis ; genetics ; Humans ; Infant ; Male

Chronic granulomatous disease (CGD) is a fatal genetic disorder in which phagocytes fail to produce antimicrobial superoxide because of NADPH oxidase deficiency. Molecular defects in CYBB gene causing X-linked CGD are responsible for about 70% of all cases. This study was done to confirm genetic defects of CYBB gene in five Korean patients who were highly suggestive of having CGD by clinical history. We performed initial screening for five unrelated Korean patients using single strand conformation polymorphism (SSCP) and then selective sequencing for the regions involving the abnormal bands. Activated NBT tests revealed that all patients were X-linked. SSCP analysis for CYBB gene showed abnormal bands in all patients. The molecular defects of five patients were as follows: c.1663insT, c.1111-1G>T, c.39_40insG, c.927delC and c.434T>C mutation. This result will help the families with prenatal diagnosis or genetic counseling.
Adult ; Child ; *Chromosomes, Human, X ; DNA Mutational Analysis ; Granulomatous Disease, Chronic/*genetics ; Human ; Infant ; Korea ; Linkage (Genetics) ; Male ; Point Mutation ; *Polymorphism, Single-Stranded Conformational ; Support, Non-U.S. Gov't

OBJECTIVETo summarize clinical and molecular features of two children with autosomal recessive chronic granulomatous disease caused by CYBA mutations.

METHODThe clinical records and CYBA mutations were reviewed for analysis of infections and inflammatory complications.

RESULTThe first case was a girl diagnosed with "liver and spleen abscess" in our hospital when she was 2.9 years old, with past history of neonatal impetigo and recurrent purulent lymphadenitis and positive family history. The results of DHR123 flow-cytometry showed that positive phagocytes after phorbol ester (PMA) stimulation was 84.63%. CYBA mutation analysis showed that she had heterozygous 35C > T, Q3X and IVS-2A > G. The second case was a boy diagnosed with "sepsis (salmonella D)" when he was 4 years old with a past history of impetigo, sepsis, perianal abscess, skin infection and positive family history. The results of flow cytometry showed that positive phagocytes after PMA stimulation was 96.13%. CYBA mutation analysis showed that he had homozygous 35C > T, Q3X and his parents were all carriers. All of them had BCG related axillary lymphnode calcification.

CONCLUSIONA22CGD cases had recurrent purulent infections (skin, lymphnode, liver and spleen, lung, blood), DHR123 flow cytometric analysis helped the diagnosis of CGD, CYBA mutation analysis ascertained the diagnosis of A22CGD.

Child, Preschool ; Chromosome Aberrations ; DNA Mutational Analysis ; Female ; Genes, Recessive ; Granulomatous Disease, Chronic ; diagnosis ; genetics ; Homozygote ; Humans ; Male ; Mutation ; NADPH Oxidases ; genetics

Chronic granulomatous disease (CGD) is a rare genetic disease, which is caused by defects in the NADPH oxidase complex (gp91phox, p22phox, p40phox, p47phox, and p67phox) of phagocytes. This defect results in impaired production of superoxide anions and other reactive oxygen species (ROS), which are necessary for killing bacterial and fungal microorganisms and leads to recurrent, life-threatening bacterial and fungal infections and granulomatous inflammation. The dihydrorhodamine (DHR) flow cytometry assay is a useful diagnostic tool for CGD that can detect absent or reduced NADPH oxidase activity in stimulated phagocytes. We report a patient with X-linked CGD carrying a novel mutation of the CYBB gene whose chimerism status following hematopoietic stem cell transplantation (HSCT) has been rapidly determined using the DHR assay. The level of DHR activity correlates well with short tandem repeat PCR analysis. Considering the advantages of this simple, rapid, and cost-effective procedure, serial measurement of DHR assay would facilitate the rapid determination of a patient's engraftment status, as a supplementary monitoring tool of chimerism status following HSCT.
Base Sequence ; *Chimerism ; DNA Mutational Analysis ; Flow Cytometry ; Granulomatous Disease, Chronic/*diagnosis/*enzymology/genetics/surgery ; *Hematopoietic Stem Cell Transplantation ; Homozygote ; Humans ; Infant, Newborn ; Male ; Membrane Glycoproteins/chemistry/*genetics ; Mutation ; NADPH Oxidase/chemistry/*genetics ; Polymerase Chain Reaction ; Rhodamines/chemistry/metabolism

Chronic granulomatous disease (CGD) is a rare hereditary disorder characterized by recurrent life-threatening bacterial and fungal infections. The underlying defect in CGD is an inability of phagocytes to produce reactive oxygen species as a result of defects in NADPH oxidase. Considering that CGD generally affects about 3-4 in 1,000,000 individuals, it is surprising that the prevalence of CGD on Jeju Island is 20.7 in 1,000,000 individuals. We performed genetic analysis on 12 patients from 10 unrelated families and found that all patients had an identical homozygous single-base substitution of C to T in exon 1 (c.7C>T) of the CYBA gene, which was expected to result in a nonsense mutation (p.Q3X). Because Jeju Island has long been a geologically isolated region, the high prevalence of CGD on Jeju Island is presumably associated with an identical mutation inherited from a common ancestor or proband.
Adolescent ; Animals ; Asian Continental Ancestry Group/*genetics ; Base Sequence ; Child ; Child, Preschool ; DNA Mutational Analysis ; Female ; Geography ; Granulomatous Disease, Chronic/*genetics ; Humans ; Infant ; Korea ; Male ; Molecular Sequence Data ; *Mutation ; NADPH Oxidase/*genetics ; Pedigree ; Young Adult

BCG Vaccine ; adverse effects ; Child ; Child, Preschool ; Genetic Diseases, X-Linked ; complications ; Granulomatous Disease, Chronic ; complications ; genetics ; Humans ; Infant ; Infant, Newborn ; Lymphadenitis ; etiology ; Male ; Membrane Glycoproteins ; genetics ; NADPH Oxidase 2 ; NADPH Oxidases ; genetics

OBJECTIVEChronic granulomatous disease (CGD) is a rare primary immunodeficiency of phagocytic oxidative bursts leading to recurrent severe bacterial and fungal infections as well as granuloma formation. There were few reports on the clinical characteristics of this disease in China. The purpose of this study was to evaluate the clinical features of 48 Chinese cases with CGD which were confirmed by clinical features, dihydrorhodamine (DHR) assay and gene mutation analysis.

METHODThe study cohort was the population of CGD patients diagnosed in Children's Hospital of Fudan University from January, 2004, to June, 2011. Cases included in our analysis were restricted to those who had complete data of the clinical symptoms and laboratory tests. The patients were followed up by outpatient visiting and telephone call regularly for 0.5 to 6 years. The history and data of physical examination and treatment of 48 cases were collected and reviewed.

RESULTAll the patients were diagnosed by DHR analysis. The age of onset of all the 48 patients were less than 6 months, including 43 male and 5 female. The mean age at diagnosis was 2.42 years; 12 patients were infants under six months, 10 were between 6 and 12 months, 9 were between 1 and 2 years, 5 patients were between 2 and 3 years, 4 were between 4 and 5 years, and 8 were between 6 and 10 years. Recurrent respiratory infection (44/48) and chronic diarrhea (31/48) were the common symptoms in all the patients, and then skin lesion (22/48), including marked reaction at BCG infected site, pustular eruption and infected skin ulcer and urinary tract infection (3/48) were also general symptoms in our study. In addition, lymphadenectasis occurred in 31 cases and 23 of them were considered to be associated with BCG vaccination. The pathogens caused the infection were mycobacteria (52.08%), fungi (43.75%) and pyogenic bacteria. Thirty-seven patients had mutations in CYBB/CYBA/NCF1/NCF2 genes. Recombinant human interferon-gamma (rhIFN-γ) plus sulfamethoxazole were used for the prevention and treatment of infection, the frequency and severity of the disease could be reduced.

CONCLUSIONThe age at onset and diagnosis of the present group of CGD was younger. Clinical symptoms were associated with recurrent mycobacterial, fungal and pyogenic bacterial infection, which involved respiratory tract, alimentary tract, skin and lymph node. rhIFN-γ partially improved the prognosis of CGD.

Bacterial Infections ; epidemiology ; etiology ; prevention & control ; Child ; Child, Preschool ; Female ; Gastrointestinal Diseases ; epidemiology ; etiology ; prevention & control ; Granulomatous Disease, Chronic ; complications ; diagnosis ; genetics ; Humans ; Infant ; Interferon-gamma ; therapeutic use ; Lung Diseases ; epidemiology ; etiology ; prevention & control ; Male ; Mutation ; Mycobacterium Infections ; epidemiology ; etiology ; prevention & control ; Recombinant Proteins ; Retrospective Studies ; Skin Diseases ; epidemiology ; etiology ; prevention & control