Clinical And Molecular Analysis Of CGD And CMC

Part one: Analysis of Clinical and Molecular Characteristics of CGDObjectives:1. To analyse the clinical and laboratory features of CGD.2. Genetic diagnosis will provided valuable evidences foridentification of X-CGD and AR-CGD, and for their suitable and correctclinical treatment.3. To detect the change of T cell subtypes including Th17, nTreg andiTreg cells of CGD.Methods:1. To analyze the clinical and laboratory data of10cases of CGDpatients.2. Neutrophil respiratory burst assay was detected by Flow cytometryin the10CGD patients（including before and after transplanting in2patients） and their family members.3. The CYBB, CYBA, NCF1, NCF2and NCF4genes of peripheralblood cells from10CGD patients（including before and after transplanting in a patient） and their family members were analyzed by PCR-directedsequencing.4. Th17, nTreg, iTreg cell numbers from CD4+T cells in PBMCs of6CGD patients（including before and after transplanting in2patients） weredeteced by Flow cytometry.Results:1. Clinical feature and laboratory examination10cases of CGD patients were all male, the awerage age of onset was4months,the average age of diagnosis was3years. Patients with CGD allsuffered severe recurrent pneumonia （100%）, tubercular infection8cases（80%）, skin and/or perianal abscesses8cases （80%）. Additionally, thepatients suffered from lymphadenitis and hepatosplenomegaly in8cases（80%）, septicemia in6cases （60%）, fungal infection in6cases （60%）.There were5observed cases of recurrent diarrhea （50%）,4cases ofintracranial infection （40%）,1case of BCG diseases （10%）,1case ofosteomyelitis （10%）, and finally1case of tympanitis （10%）. All patientshad dystrophia, growth and developmental retardation. Two patients had aprevious family history. Two cases were dead and two cases had stem celltransplantation.10CGD patients all have elevated WBC, mainly in neutral. SerumIgG and some IgE were increased. The NBT were0%and the neutrophiloxidative fuction were all significantly lowered. Serum IgG and WBC were decreased, the NBT and neutrophil oxidative fuction became normal after24days of stem cell transplantion in P2and P4. The pathogens maininclude staphylococcus aureus and candida albicans. Imaging showedhigh-density nodules in8cases, bronchiectasia and pulmonary fibrosis in1case, and hepatosplenomegaly in8cases.2. Gene analysisCYBB gene mutations were found in eight X-CGD patients andCYBA gene mutation was found in an AR-CGD patient. There was no genemutation found in the other patient. The candidate genes included CYBB,CYBA, NCF1, NCF2and NCF4. The CYBB gene mutations were foundincluding2deletion mutations which were found in3patients （c.1122delG;XK, CYBB and DYNLT3gene klenow fragment deletion）,1missensemutation （c.1082G>T） and4splicing errors（c.46-2A>G; c.674+1delG;c.338₆74del, c.484₆74del; c.484₈04del）. A CYBA gene missensemutation（c.152T>G） was found. The mutations had not been reportedbefore except one（c.1082G>T）.3. Th17, nTreg, iTreg cell numbers from CD4+T cells in PBMCsThe proportion of Th17cells in CD4+T cells was tested in6patients（0.22±0.15%） and was found to be significantly lower compared with6healthy controls（1.02±0.20%）. Th17cell numbers in patients2and4demonstrated no significant change before and after transplantation. Theproportion of nTreg and iTreg cells in CD4+T cells was tested in6 patients（3.48±2.53%,7.46±6.02%）， almost normal compared with6healthy controls（3.17±0.96%,8.96±3.19%）. The nTreg cells before andafter transplantation in P2and P4were6.83%,5.99%and3.31%,4.94%respectively. The iTreg cells before and after transplantation in P2and P4were4.00%,4.38%and9.29%,12.68%respectively. There were slightdecreasing in nTreg and significantly increased in iTreg after transpltation.Conclusions: CGD patients in this group which had an early onsethad severe clinical manifestation. The main infection pathogens arebacterium, fungus and mycobacterium especially. All patients hadhypergammaglobulinemia and elevated leukocyte, mainly in neutrophils.Neutrophil respiratory burst test and gene analysis may be used to thediagnose patient and fetus with CGD to make early diagnosis and guidetreatment. The proportion of Th17cells was significantly lower comparedwith the healthy controls. The proportions of nTreg and iTreg cells werealmost normal compared with healthy control. Part two: Prenatal diagnosis for high-risk fetuses with chronicgranulomatous diseaseObjectives:1. To investigate the value of gene analysis of amniotic fluid of exfoliated cells in prenatal diagnosis of high-risk fetuses with CGD．2. To investigate the value of the neutrophil respiratory burst assay andgene analysis of embryo cord blood in prenatal diagnosis of high-riskfetuses with CGD.Methods:1. Six samples of amniotic fluid gotten by amniocentesis werecollected from6high-risk fetuses in the5pedigrees. CYBB and CYBAgenes were amplified by polymerase chain reaction （PCR） from RNA andDNA of amniotic fluid exfoliated cells and sequencing was performeddirectly on the PCR products.2. Embryo blood sample were collected from two high-risk fetuses bypercutaneous umbilical blood sampling. Neutrophil respiratory burst assaywas detected by Flow cytometry and the genes above were analysed.Results:1. The result of CYBB, CYBA gene analysis of amniotic fluidexfoliated cells of the six fetuses showed that a female fetus was normal, afemale fetal carrier was unidentified, two male fetuses were X-CGDpatients, a male fetus was suspicious X-CGD patient in4X-CGD pedigrees,and a female fetus was suspicious AR-CGD patient in an AR-CGDpedigree.2. The neutrophil respiratory burst function detected of embryo cordblood samples of the two suspicious CGD fetuses was shown to be very low, and the genes analysis of above two embryo cord blood samplesidentified an X-CGD male fetus and an AR-CGD female fetus.Conclusios: Gene analysis of amniotic fluid exfoliated cells andembryo cord blood cells, neutrophil respiratory burst assay of embryo cordblood can provide reliable prenatal diagnosis for high-risk fetuses withCGD. Part three: Clinical and Molecular Characteristics of CMCObjectives:1. To investigate the clinical features of a CMC patient.2. Genetic diagnosis will provided valuable evidences foridentification of the patient and genetic counseling in his families.3. To analyze Th17cells count in peripheral blood of the patient.4. Spectratyping analysis of T-cell receptor β-chain variable region（TCRVβ） gene rearrangement in the patient.Methods:Analyze the clinical characteristic, STAT1gene mutation, Th17cellnumbers from CD4+T cells in PBMCs and TCRVβgene rearrangementpolymorphism of a CMC patient to improve the understanding of the desease in pediatrician.Results:1. Clinical featureThe patient was a6year10months old boy who presented withpersistent and recurrent oral mucosa, nail and cutaneous candidosis fromthe infancy, with a history of recurrent respiratory tract infection, otitismedia and failure to thrive. He had white curd all over the oral mucosa. Alot of white secretions were found by the ear endoscope. Oral and earcandida infections were confirmed in microscopic examination of fungus.Bacterial culture of sputum specimens permitted the growth ofStaphylococcus aureus and streptococcus pneumoniae. Chest CT of thepatient showed diffuse anomalism increased density focuses, edgeunsharpness, density not well-distributed.2. Gene analysisA novel heterozygous missense mutation was identified in exon4ofSTAT1gene （g.5280A>T, p.N89Y）. Further study of family membersrevealed that his parents did not have the mutation.3. Th17cell numbers from CD4+T cells of peripheral blood PBMCsThe proportion of Th17cells in CD4+T cells in the patient was0.05%,which was less than that in normal control （0.87%）.4. Spectratyping analysis of TCRVβThe repertoire of TCRVβgene rearrangement polymorphism in the patient was partial restricted.Conclusions: The clinical manifestations of CMC were characterizedby the history of persistent and recurrent candidiasis at all body locations.We summarized the clinical, immunologic, and molecular characteristics ofa patient with AD-CMC caused by a novel mutation in the STAT1gene.The inability to clear C.albicans in CMC patients could be due to a defectin the immune response of IL-17-producing T cells. Partial restrictedrepertoire of TCRVβcould explain the abnormal development of the T cell.