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

PART ONE: OPTIMIZATION OF SCREENING METHODS FOR MENDELIAN SUSCEPTIBILITY TO MYCOBACTERIAL DISEASEObjective: Improved detection of IL-12/IFN-g axis function by Q-RT-PCR and protein expression of IFN-gR1 and IL-12Rb1 by flow cytometry, improved sensitivity, accuracy, stability, and shorten the test time.Methods: 1.Venous blood from a healthy person was divided into four groups: with medium alone, with BCG, with BCG plus IL-12p70, and with BCG plus IFN-γ. All total RNA of these groups was respectively extracted after 24, 36, 48 hours, and the m RNA expression level of IFN-γ and IL-12 B was detected by Q-RT-PCR. Repeat 3 times by different sources of whole blood, and select the optimum stimulation training time. 2. PBMC was extracted from the blood of a healthy person, and was stimulated in different concentrations of PHA(7.5 mg/ml, 10 mg/ml) and after different time(48h, 72 h, 88h), then to detect IFN-gR1 and IL-12Rb1 by flow cytometry. Repeat 3 times by different sources of whole blood to select the optimum concentration of PHA and stimulation training time.Results: 1. The best incubation time of the IL-12/IFN-g axis test was 36 hours. 2. The stimulation optimal concentration of PHA in the test of detecting IFN-gR1 and IL-12Rb1 protein expressions was 10 mg/ml, and the best incubation time was 72 hours.Conclusions: The detection of IL-12/IFN-g function axis by Q-RT-PCR and protein expression of IFN-gR1 and IL-12Rb1 by flow cytometry had been established before, and then based on the experiment, we optimized the test furtherly, selected the best incentive condition and cell culture time, improves the accuracy, sensitivity and stability, and shortened the time for clinical information.PART TWO: APPLICATION OF SCREENING METHODS FOR MENDELIAN SUSCEPTIBILITY TO MYCOBACTERIAL DISEASEObjective: To screen 23 new cases who were suspected for MSMD; To follow-up patients with abnormal test result before the optimization of MSMD(Mendelian susceptibility to mycobacterial disease) screening test; To sequence the related genes of patients with abnormal results.Methods: 1. Detected 23 new cases’ s IL-12/IFN-g axis function by Q-RT-PCR and protein expression of IFN-gR1 and IL-12Rb1 by flow cytometry.2. Follow-up patients with abnormal test result before the optimization of MSMD screening test, and review the result after obtaining permission from the parents. 3. Collected the clinical data, and combined with the experimental results of 9 new cases screened by the optimization method, and one undiagnosed but highly suspected MSMD case who screened by the method before optimization, then analyze the related genes.Results: 1. There were 9 children with abnormal test results in the 23 new clinical MSMD suspected cases, and the four women and five men on average onset age of 5.41 m. 6 cases with adverse reactions after BCG vaccination, in the other three cases, one was not vaccine BCG, and the other two were without BCG vaccine adverse reactions. All of them were girls who had a history of recurrent fever, infections and ectoderm dysplasia phenotype. 2. There were 6 patients with abnormal test results before optimization screening methods of MSMD, and one case died, one case lost to follow-up, the rest 4 cases were better recovery, but only one case came to review, the test results of both IL-12/IFN-g axis function and protein expression of IFN-gR1 and IL-12Rb1 were normal. 3. Analyze related genes of the 9 cases of 23 new clinical MSMD suspected cases with abnormal screening results of MSMD, and the dead one of 6 follow-up cases. We found one case with a new NEMO novel missense mutation, and each one with a fragment missing of CYBB gene, IL12RB1 gene and IL12 B gene.Conclusions: Compared with before, the optimized screening method of MSMD can provide a basis for diagnosis in patients more quickly, and the results were more stable and accurate. Clinically suspected MSMD children, about 90% had BCG infection, and about 40% had IL-12/IFN-g axis function damage, but there were still 60% not to find disease genes, which suggests there may be more virulence genes were not found.PART THREE: A NOVEL MISSENSE MUTATION OF NEMO GENE IN A BOY WITH ECTODERMAL DYSPLASIA AND IMMUNODEFICIENCY IN CHINAObjective: To explore the clinical features and NEMO gene mutation in a boy with ectodermal dysplasia and immunodeficiency(EDA-ID), and improve the clinicians’ early recognition.Methods: According to the patient’s clinical presentation and family history, we detected the integrity of the IL-12/IFN-γ axis by Q-RT-PCR after ruling out the common primary immunodeficiency disease(PID), then the NEMO gene was sequenced.Results: The patient appeared with fever repeatedly, and had a pale skin, sparse hair, mild frontal bossing, fungus and BCG infections. The X-ray of head revealed no obvious tooth germ. The expression of IL-12 B m RNA by whole blood cells from the patient reduced after stimulation with BCG plus IFN-γ than BCG alone(P(27)0.05). NEMO gene sequencing analysis found a c.1241T(29)A substitution mutation in exon 10, predicting a valine to aspartic acid missense mutation at amino acid position 414(p.V414D). The mother and one of her sisters had heterozygous mutations at the same position.Conclusions: Male children with BCG infections and recurrent fever should be considered a mutation of the NEMO gene, especially when there are some ectoderm dysplasia phenotype, such as a pale and dry skin without sweat, sparse hair, no teeth. Matrilineal family history of incontinentia pigmenti is helpful to diagnosis. EDA-ID can be diagnosed early combining the integrity of IL-12/IFN-γ axis testing and genetic analysis.