Establishment And Application Of Screening Methods For Mendelian Susceptibility To Mycobacterial Disease

PART ONE: ESTABLISHMENT OF SCREENINGMETHODS FOR MENDELIAN SUSCEPTIBILITY TOMYCOBACTERIAL DISEASEObjective:1. To establish the function test to detect the integrity ofIL-12/23-IFN-γ pathway by Q-RT-PCR2. To establish the method to detect IL-12RB1and1IFN-γR1by flowcytometryMethods:1. To detect IFN-γ mRNA levels in whole blood diluted withRPMI1640by Q-RT-PCR, after stimulation with different concentrations ofBCG (Blank control,2.5ug/ml,0.25ug/ml,0.0250ug/ml,0.0025ug/ml) for48h, and repeat three times with different health volunteers2. To detect IFN-γ mRNA levels in whole blood diluted withRPMI1640by Q-RT-PCR, after stimulation with BCG plus differentconcentrations of IL-12P70（Blank control,40ng/ml,20ng/ml,10ng/ml， 5ng/ml）for48h, and repeat three times with different health volunteers3. To detect IL-12B mRNA levels in whole blood diluted withRPMI1640by Q-RT-PCR, after stimulation with BCG plus differentconcentrations of IFN-γ (Blank control,10000U/ml,5000U/ml,2500U/ml,1250U/ml) for48h, and repeat three times with different health volunteers4. To detect IL-12RB1and IFN-γR1by flow cytometry, with inducedby PHA (5ug/ml) for24h,48h, and88h, respectivelyResults:1. The optimal stimulation conditions of the function test ofIL-12/23-IFN-γ pathway were as follows:1) Blank control;2）live BCG (0.25ug/ml) alone;3）BCG (0.25ug/ml)plus IL-12P70(20ng/ml);4) BCG (0.25ug/ml) plus IFN-γ (2500U/ml)2. Stimulated with PHA (5ug/ml) for88h is the best condition to detectIL-12RB1and1IFN-γR1by flow cytometryConclusion:We established methods to detect the integrity of IL-12/23-IFN-γpathway by Q-RT-PCR and IL-12RB1and1IFN-γR1by Flow cytometry.Combination with these two methods may be effective for the diagnosis ofMSMD quickly. PART TWO：SCREENING OF CHILDREN WITHABNORMAL RESPONSE AFTER BCG VACATION FORMENDELIAN SUSCEPTIBILITY TO MYCOBACTERIALDISEASEObjective:To screen12patients with abnormal responses after BCG vaccinationto determine whether they presented with Mendelian Susceptibility toMycobacterial Disease or not.Methods:1. To collect clinical information and laboratory tests of12patientswith abnormal responses after BCG vaccination.2. The integrity of IL-12/23-IFN-γ pathway was tested in12patientsand healthy controls by Q-RT-PCR.3. The expression of IL-12RB1and IFN-γR1were analyzed by flowcytometry in9patients.4. IL12RB1and IFNGR1genes were determined by RT-PCR/PCR-direct sequencing.Results:1. Clinical features and laboratory tests12patients including seven males and five females were allcharacterized by lymphadenitis after BCG vaccination,in addition to this，9cases and7cases had other medical and family histories, respectively. The mean age of onset was6.03months, while the mean age of screening was19.77months. To date,2cases (patients3and9) died.Lymph node biopsy results showed infections with tuberculosis in10cases. Chest CT showed abnormal in5cases.2. The integrity of IL-12/23-IFN-γ pathway was tested in12patients.There were abnormal in patients1,3,9and10.3. We did not detect the expression of IL-12RB1in patients4and9.4. IFNGR1and STAT1gene mutations were found in patients3and10,respectively.Conclusions:In our cohort,12patients were all characterized by lymphadenitis afterBCG vaccination, especially occurring in ipsilateral axillary lymph node.Patient3was diagnosed as MSMD caused by IFNGR1gene mutation.Patient10was diagnosed as CMC because of gain-of-function mutation inthe STAT1. PART THREE: CLINICAL FEATURES, FUNCTION ANDMOLECULAR ANALYSIS OF IFN-Γ RECEPTOR1DEFICIENCYObjective:To explore the clinical features, detect the function of the IL-12/23-IFN-γ axis and identify the IFNGR1gene mutation in a child withsuspected Mendelian susceptibility to mycobacterial disease (MSMD), toimprove the pediatricians’ understanding of this disease.Methods:Firstly, we ruled out the common primary immunodeficiency disease(PID) based on the clinical manifestation and screening tests of immunefunction, and then detected the integrity of the IL-12/23-IFN-γ axis byQ-RT-PCR. IFNGR1gene was sequenced according to the results ofQ-RT-PCR.Results:1. A11-month-old girl who presented with a history of4months ofanemia and cough was admitted to the Children’s Hospital of ChongqingMedical University. Anemia is a remarkable clinical manifestation in thischild, but she had no symptom of internal bleeding and hemolysis. The childsuffered from disseminated BCG disease. X-ray of the limbs long bone andCT chest and abdomen showed multiple osteolytic processes.2. All of immunoglobulin levels, lymphocyte sub-classification, NBT,CD18and DHR flow cytometry assay were normal.3. The production of IL-12B in response to BCG plus IFN-γ wasreduced compared with stimulation by BCG alone, but the healthy controlincreased. 4. A heterozygous deletion mutation was found in exon6(c.818-821delTTAA, p.Asn274fsX276) in IFNGR1gene of the patient. IFNGR1geneswere normal in the parents.Conclusions:Detection the function of IL-12/23-IFN-γ axis is an effective tool forthe screening of MSMD quickly. IFNGR1genetic mutation is responsiblefor BCG infections and multi-system lesions in this child.