| PART ONE: MECHANISM OF CHANGES OF MEMORY B CELLS IN PATIENTS WITH CHRONIC GRANULOMATOUS DISEASEObjective: To investigate the changes of number of memory B cell(Bm)in peripheral blood of patients with chronic granulomatous disease,and to explore the reasons for the changes.Methods: Flow cytometry was used to detect the percentage of Bm,GC-Bm(Germinal center dependent memory B cell),GC-inBm(Germinal center independent memory B cell),PC(Plasma cell),Na?ve B,Th(T helper cell),Tfh(T follicular helper cell)and the expression of ICOS and PD-1 on the surface of Tfh in 15 CGD patients and 15 healthy controls.Flow cytometry was used to detect the percentage of CD40 on Bm surface and CD40 L on Tfh surface at different stimulation time in CGD patients and healthy controls.Results: The proportion of Bm in CGD patients(1.21%+0.91%)was significantly lower than that in the control group(2.79%+0.85%)(P<0.0001);the proportion of Nave B in CGD group(79.63%+12.68%)was significantly higher than that in the control group(59.06%+13.57%)(P=0.0002);and the proportion of PC in CGD group(1.45%+1.56%)was significantly lower than that in the control group(3.67%+2.11%)(P=0.0029).The proportion of Tfh in CGD patients(2.84%+1.9%)was significantly lower than that in the control group(4.58%+1.86%)(P=0.0169).There was no significant difference in Th ratio,ICOS and PD-1 molecule ratio on Tfh surface.There was no significant difference in CD40 on the surface of B cells.The percentage of CD40 L on Tfh cell surface of CGD patients(1.97%+0.82%)was significantly lower than that of control group(4.96%+1.39%)(P=0.0011);after 4 hours of stimulation,the percentage of CD40 L on Tfh cell surface of CGD patients(18.32%+11.72%)was significantly lower than that of control group(36.93%+15.34%)(P=0.0399);after 6 hours of stimulation,the difference was not significant.Conclusion: The process of differentiation of Na?ve B into Bm and PC in CGD patients has some defects,which leads to the increase of Na?ve B and the decrease of Bm and PC in patients’ peripheral blood,which may be one of the causes of repeated infection.Defects in the differentiation process of Na?ve B may be related to the decrease of Tfh cell number and CD40 L expression on the surface of CGD patients.PART TWO: X-LINKED CHRONIC GRANULOMATOUS DISEASE IN A FEMALE PATIENTObjective: Here reports the respiratory burst function and protein expression of neutrophils and the CYBB gene characteristics before and after delivery of a rare female X-linked chronic granulomatous disease infant patient,which enriches the understanding of CGD and warns the prenatal diagnosis.Methods: Gene sequencing was used to detect the related genes in amniotic fluid cells and peripheral blood cells of patient and her parents.Flow cytometry was used to detect the respiratory burst function(DHR-1,2,3)and the expression of gp91 phox protein of neutrophils in peripheral blood of patient and parents.X chromosome inactivation was detected in patient and parents.Results: Clinical data: The patient,female infant,1 year and 11 months old,newborns with ABO hemolysis and eczema after birth,BCG vaccination at the age of 1 month,ulceration at the vaccination site at the age of 2 months,and scar formation after 20 days.Perianal abscess was the first symptom at 2 months and 21 days of age,and axillary lymph node suppuration occurred once at 6 months,and suffered from pneumonia 3 times and right cervical lymph node enlargement 1 time.The two twin brothers were both CGD patients and their mothers were carriers.CYBB gene analysis: g DNA level of amniotic fluid cells at 20 weeks gestational age showed a heterozygous deletion mutation at exon9 c.1123 del G.After birth,the g DNA level of peripheral blood showed a heterozygous deletion mutation,while the cDNA level showed a homozygous deletion mutation at exon9 c.1123 del G,which resulted in the protein change of p.Glu375 Serfs X11;the g DNA and cDNA level of peripheral blood of the mother were heterozygous mutation;the father had no mutation.Respiratory burst test: SI of the patient was 0.65,the mother showed double peaks after stimulation,the SI was 10.86,and the father’s SI was 134.93.The gp91 phox protein: no expression in patient,partial expression in mother and complete expression in father.X chromosome inactivation: the paternal X chromosome inactivation rate of patient was 99.5% and the mother was 76.1%.Conclusion: Because of the possibility of normal X chromosome inactivation in female carriers,the g DNA and cDNA of fetuses should be analyzed during prenatal diagnosis,and the respiratory burst function and the expression of gp91 phox protein of neutrophils in umbilical cord blood should be examined in female CYBB heterozygous mutation fetuses.PART THREE: CLINICAL MANIFESTATIONS AND MOLECULAR CHARACTERISTICS OF PATIENTS WITH CHRONIC GRANULOMATOUS DISEASEObjective: To investigate the clinical manifestations,laboratory tests,etiology and gene mutations of 114 patients with chronic granulomatous disease(CGD).Methods: The clinical data and laboratory examination results of 114 patients with CGD admitted to Children’s Hospital of Chongqing Medical University from January 2003 to October 2018 were collected and analyzed.Flow cytometry was used to detect the respiratory burst function of neutrophils in suspected patients,and SI was calculated.The CGD-related genes of patients were detected and analyzed.Results: Patients: Among 114 cases,109 were male,5 were female and came from 109 families.62(54.4%)patients came from southwestern of China,and 49 patients(42.9%)had family history.The age of onset ranged from 2 days to 72 months.The average age of onset was 4 months,and the median was 1 month.The age of diagnosis ranged from 10 days to 172 months,with an average age of 23 months and a median age of 12 months.A total of 33 patients died,the average age of death was 40 months(range from 6 to 120 months),and the median age of death was 31 months.29 patients received hematopoietic stem cell transplantation(HSCT),2 of them died and 1 failed.Infections: 114 cases had recurrent fever(100%),105 cases had pneumonia(92.1%),84 cases had abscess(73.7%),67 cases had lymphadenitis(58.8%),67 cases had hepatosplenomegaly(58.8%),67 cases had tuberculosis infection(58.8%),54 cases had BCG disease(55.1%),52 cases had diarrhea(45.6%),48 cases had fungal infection(42.1%),35 had sepsis(30.7%),19 had thrush(16.7%),8 had urinary tract infection(7.0%)and 5 had osteomyelitis(4.4%).Laboratory tests: The level of peripheral white blood cells and CRP in most patients was higher than normal range.Immunoglobulin was normal or increased,and lymphocyte classification showed no significant change.NBT ranges from 0 to 6 with an average of 1 and a median of 0.DHR-1,2,3 was carried out in 113 children with SI ranging from 0.1 to 15,with an average of 1.67 and a median of 1.31,of which 95(84.1%)were lower than 2.32 patients found evidence of fungal infection,mainly were Candida albicans and Aspergillus fumigatus.37 patients found evidence of bacterial infection,mainly were Klebsiella pneumoniae,Staphylococcus aureus,Escherichia coli,Moraxella catarrhalis.Gene mutation analysis and prenatal diagnosis: 95 cases were CYBB gene mutation,among which splicing mutation(30,31.6%)was the most common,followed by deletion mutation(27,28.4%),nonsense mutation(18,18.9%)and missense mutation(18,18.9%).8 cases were CYBA,3 cases were NCF1 and 2 cases were NCF2 gene mutations.In 6 cases,no mutation sites were found in g DNA and cDNA.4 site with high mutation frequency were found,CYBB: 4 cases in c.252 G > A,3 cases in c.676 C > T,4 cases in c.868 C > T,and CYBA: 3 cases in c.152 T > A.Prenatal amniotic fluid gene testing screened 15 cases,including 7 cases,5 healthy children and 3 carriers.Conclusion: CGD should be suspected in children within one year old with severe pneumonia,abscess,lymphadenitis,tuberculosis,BCG disease and recurrent diarrhea.In this study,the majority of CGD patients have SI less than 2,the main mutation gene is CYBB gene,and the main mutation type is splicing mutation.Flow cytometry can be used to detect the respiratory burst function of neutrophils for rapid diagnosis of CGD,and gene detection can further confirm the diagnosis. |
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