Vitamin D supplementation reduced blood inflammatory cytokines expression and improved graft function in kidney transplant recipients.

Background: Chronic allograft dysfunction(CAD) is the leading cause of graft loss in kidney transplant recipients (KTRs). Inflammatory process is believed to be one of the major contributors to CAD. The aim of this study is to explore the anti-inflammatory effect of vitamin D (VD) supplementation in KTRs and its role in the graft function improvement(protection). Methods: A retrospective cohort of 39 KTRs with chronic antibody mediated rejection(CAMR)or stable renal function and a prospective cohort of 42 KTRs treated or untreated with VD were enrolled. Serum levels of vitamin D metabolism and serum inflammatory cytokines, renal graft function, and routine blood biomarkers were tested and dynamically tracked within 12 months post-transplant. Results: Compared with the stable group, the CAMR group exhibited significantly elevated serum levels of inflammatory cytokines IL-1ß, IFN-γ, IL-2, IL-10, IP-10, and HMGB1 (P <0.05). The supplementation of vitamin D effectively increased the serum concentration of vitamin D in kidney transplant recipients (KTRs) in the treated group. During the course of treatment, the treated group exhibited a gradual increase in eGFR levels, which were significantly higher than those observed in the untreated group at 12 months post-transplant (p<0.05). Notably, as eGFR improved, there was a significant decrease in levels of IL-1ß, IFN-γ, IL-2, IL-10, IP-10 and HMGB1 in the treated group compared to the untreated group (P<0.05). Conclusion: This study confirmed that immune-inflammation is a crucial factor in the development of CAD in KTRs.VD deficiency impairs its anti-inflammatory activity. By assisting in the regulation of excessive immune inflammation and restoration of immune homeostasis, effective VD supplementation contributes to protection and maintenance of graft function in KTRs.

[Gene editing for the treatment of primary immunodeficiency disease].

Gene editing is an advanced technique based on artificial nucleases and can precisely modify genome sequences. It has shown great application prospects in the field of medicine and has provided a new precision therapy for diseases. Primary immunodeficiency disease is a group of diseases caused by single gene mutation and characterized by recurrent and refractory infections, with an extremely high mortality rate. The application of gene editing has brought hope for curing these diseases. This article reviews the development of gene editing technology and briefly introduces the research and application of gene editing technology in primary immunodeficiency disease.

Dynamics of NK, CD8 and Tfh cell mediated the production of cytokines and antiviral antibodies in Chinese patients with moderate COVID-19.

Recent studies have demonstrated a marked decrease in peripheral lymphocyte levels in patients with coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Few studies have focused on the changes of NK, T- and B-cell subsets, inflammatory cytokines and virus-specific antibodies in patients with moderate COVID-19. A total of 11 RT-PCR-confirmed convalescent patients with COVID-19 and 11 patients with non-SARS-CoV-2 pneumonia (control patients) were enrolled in this study. NK, CD8+ T, CD4+ T, Tfh-like and B-cell subsets were analysed using flow cytometry. Cytokines and SARS-CoV-2-specific antibodies were analysed using an electrochemiluminescence immunoassay. NK cell counts were significantly higher in patients with COVID-19 than in control patients (P = 0.017). Effector memory CD8+ T-cell counts significantly increased in patients with COVID-19 during a convalescent period of 1 week (P = 0.041). TIM-3+ Tfh-like cell and CD226+ Tfh-like cell counts significantly increased (P = 0.027) and decreased (P = 0.022), respectively, during the same period. Moreover, ICOS+ Tfh-like cell counts tended to decrease (P = 0.074). No abnormal increase in cytokine levels was observed. The high expression of NK cells is important in innate immune response against SARS-CoV-2. The increase in effector memory CD8+ T-cell counts, the up-regulation of inhibitory molecules and the down-regulation of active molecules on CD4+ T cells and Tfh-like cells in patients with COVID-19 would benefit the maintenance of balanced cellular and humoural immune responses, may prevent the development of severe cases and contribute to the recovery of patients with COVID-19.

Assessment of serum Tim-3 and Gal-9 levels in predicting the risk of infection after kidney transplantation.

Infection remains a major cause of morbidity and mortality after kidney transplantation (KT). Reliable biomarkers to predict post-transplant infection are lacking. We investigated the predictive performance of pre- and post-transplant levels of T-cell immunoglobulin and mucin domain-3 (Tim-3) and Galectin-9 (Gal-9), two pleiotropic immunomodulatory molecules, in early identification of infection. Serum Tim-3 and Gal-9 were paired measured before and 30 days after transplantation (PTD 30) in 95 KT recipients (KTRs). The decline rates of Tim-3 and Gal-9 were calculated relative to pre-transplant levels. KTRs with infection history had significantly higher levels of PTD 30 Tim-3 and Gal-9, and slower decrease rates of Gal-9 compared to non-infected recipients, while no difference was observed between two groups regarding pre-transplant levels. The AUCs for predicting 1-year post-transplant infection were 0.653 and 0.711 for post-transplant Tim-3 and Gal-9, 0.664 and 0.670 for relative Tim-3 and Gal-9, respectively. After adjusting for potential confounders, PTD 30 Tim-3, Gal-9 and relative Gal-9 remained as independent risk factors for post-transplant infection. Our results suggested that PTD 30 Tim-3 and Gal-9 and relative decrease of Gal-9 were promising predictors for identifying KTRs with high risk of infection, while pre-transplant Tim-3 and Gal-9 showed no predictive power to infection.

Wiskott-Aldrich syndrome protein may be critical for CD8+ T cell function following MCMV infection.

Wiskott-Aldrich syndrome (WAS) patients are characterized by immunodeficiency and viral infections. T cells derived from WAS patients and WAS protein (WASP)-deficient mice have various defects. However, whether WASP plays a role in immune control of cytomegalovirus (CMV) infection remains unclear. We analyzed the distribution of CD8+ T subsets and the pathological damage to various organs and tissues in MCMV infected Was knockout (KO) mice. A relatively high number of MCMV-specific cytotoxic T cells (CTLs) were observed in the spleen of Was KO mice. In MCMV infected Was KO mice, the late differentiated CD8+ T subset (CD27-CD28-) decreased in lungs, compared with those in the spleen and peripheral blood. Additionally, we found that the most severe pathological lesions occurred in the lungs, the main target organ of MCMV infection. By stimulating the spleen-derived CD8+ T lymphocytes of Was KO mice, we found that IL-2 and granzyme B production declined compared with that in wild- type mice. Moreover, the number of apoptotic CD8+ T cells increased in Was KO mice compared with the number in wild-type mice. Therefore, our results demonstrate that WASP may be involved in regulating cytotoxic function and apoptosis in CD8+ T cells following MCMV infection, which is supported by the distribution and memory compartment of MCMV-specific T cells in MCMV infected WAS mice.

Enhanced IL-6/phosphorylated STAT3 signaling is related to the imbalance of circulating T follicular helper/T follicular regulatory cells in patients with rheumatoid arthritis.

BACKGROUND: Follicular helper T (Tfh) cells are specialized in helping B lymphocytes, which play a central role in autoimmune diseases that have a major B cell component, such as in rheumatoid arthritis (RA). Follicular regulatory T (Tfr) cells control the over-activation of Tfh and B cells in germinal centers. Dysregulation of Tfh cells and Tfr cells has been reported to be involved in the pathogenesis of some autoimmune diseases. However, the balance of Tfh and Tfr cells, and their roles in the development and progression of RA are still not clear. METHODS: In this study, we enrolled 44 patients with RA (20 patients with active RA and 24 patients with inactive RA) and 20 healthy controls, and analyzed the frequencies of circulating Tfh and Tfr cells, expression of programmed death-1 (PD-1), inducible co-stimulator (ICOS), intracellular IL-21, and pSTAT3 in Tfh cells, and serum levels of IL-6. The correlation among these parameters and that of Tfh or Tfr cells with disease activity were also analyzed. RESULTS: Patients with RA (especially active RA) had higher frequencies of Tfh cells, but lower percentages of Tfr cells, thereby resulting in elevated ratios of Tfh/Tfr. Expression levels of PD-1 and IL-21 in Tfh cells were higher in patients with RA than in healthy subjects, while no difference in ICOS expression was observed between patients and controls. Both pSTAT3 expression and serum IL-6 levels increased in patients with RA, and positive correlation between them was observed. Additionally, pSTAT3 expression was positively correlated with Tfh cell frequency. The Disease Activity Score in 28 joints based on C-reactive protein (DAS28-CRP) was negatively correlated with Tfr cell frequency, but was positively correlated with both Tfh/Tfr ratio and PD-1 expression. CONCLUSIONS: Results demonstrated that enhanced IL-6/pSTAT3 signaling may contribute to promotion of Tfh cells, consequently skewing the ratio of Tfh to Tfr cells, which may be crucial for disease progression in RA.

The influence of living donor SHROOM3 and ABCB1 genetic variants on renal function after kidney transplantation.

OBJECTIVE: A genome-wide association study has identified several gene polymorphisms associated with loss of renal function. The effect of these variants on renal function in kidney transplant recipients receiving immunosuppressive treatment is unknown. MATERIALS AND METHODS: A cohort of 189 kidney transplant recipients and their living donors were recruited from West China Hospital of Sichuan University, on whom we assessed the association of five single nucleotide polymorphisms with renal function after kidney transplantation. RESULTS: Glomerular filtration rate estimated by serum creatinine was significantly higher in recipients carrying allograft with the A allele at rs17319721 in SHROOM3 (shroom family member 3) than those in the group with the GG genotype from month 1 to month 6 after transplantation (P=0.020). Covariate adjustment analysis showed that the variant at rs17319721 in SHROOM3 was an independent risk factor for renal dysfunction after the first month after transplantation (P=0.022). The estimated glomerular filtration rate was the lowest in recipients with allograft carrying both the A allele at rs17319721 in SHROOM3 and the CC genotype at rs1045642 in ABCB1 (P<0.05). CONCLUSION: The genetic variants in SHROOM3 and ABCB1 in donors were associated closely with renal function after kidney transplantation.

Donor ABCB1 3435 C>T genetic polymorphisms influence early renal function in kidney transplant recipients treated with tacrolimus.

AIM: Determine the effect of genetic variants of donors and recipients on early renal function and tacrolimus pharmacokinetics in kidney transplant recipients. PATIENTS & METHODS: We measured CYP3A5 (6986A>G), ABCB1 (3435C>T, 2677G>T/A, 1236C>T) genetic polymorphisms in 120 renal transplant recipients and donors by high-resolution melting curve analysis. The renal function and tacrolimus trough concentrations were analyzed. RESULTS: The genotype of CYP3A5 (6986A>G) in recipients showed strong influence on tacrolimus pharmacokinetics. The ABCB1 3435 CC genotype in donor was significantly associated with lower estimated glomerular filtration in recipients within 1 month (p < 0.05) and correlated with delayed creatinine recovery (p = 0.007). CONCLUSIONS: ABCB1 3435 CC genotype in donor influences early renal function and creatinine recovery in tacrolimus-treated kidney transplant recipients.

[Genotype of Thalassemia in Han Chinese and Tibetans in Sichuan Province,China.]

OBJECTIVES: To investigate the carrying rates and genotype distribution of thalassemia gene in Han people and Tibetans in Sichuan district. METHODS: A total of 1 147 Han adults and 613 adult Tibetans were included in this study.Hematological parameters were measured with Sysmex XE-2100 automatic blood cell analyzer.Alpha thalassemia and beta thalassemia gene analyses were further performed on samples with a mean corpuscular volume (MCV) <85 fL and a mean corpuscular hemoglobin (MCH) <27 pg.Multiplex ligation-dependent probe amplification (MLPA) and reverse dot blot assays were used for detecting deletional mutations of α globin gene and non-deletional mutations of α globin and ß globin genes,respectively. RESULTS: About 1.48% (17/1 147) Han people carried alpha thalassemia gene,with --SEA/αα as the most common genotype; 1.39% (16/1 147) carried beta thalassemia gene,with CD17 and IVS-2-654 as the most common genotype.There were 2 cases with both alpha and beta thalassemia.Low MCH (<27 pg) was found in all 33 cases with positive thalassemia genes.However,5 people with positive thalassemia genes had higher than 80 fL MCV,with the highest reaching 83.7 fL.Out of 613 Tibetans,only one was found to have positive thalassemia genes. CONCLUSIONS: Sichuan Han population carry a high level of thalassemia genes,with various genotypes and pathogenic gene mutation types.Han people with < 84 fL MCV and <27 pg MCH were recommended for thalassemia gene screening.Tibetans were not recommended for routine screening of thalassemia.

[MonoMAC syndrome].

MonoMAC syndrome is a newly discovered immune deficiency syndrome caused by GATA-2 mutation, which is an autosomal dominant genetic disease. MonoMAC syndrome has typical immune cell abnormalities, with severe infection and is prone to develop into a hematological disease. Therapeutics for this disease mainly relies on symptomatic treatment and hematopoietic stem cell transplantation. In this paper, the research advances in clinical manifestations, laboratory tests, pathogenesis, diagnosis and treatment of MonoMAC syndrome are reviewed.

Clinical and molecular characteristics of Chinese patients with X-linked lymphoproliferative syndrome type 1.

BACKGROUND: X-linked lymphoproliferative syndrome type 1 (XLP1) is a rare inherited, life-threatening immunodeficiency disorder caused by mutations in SH2D1A gene. It affect approximately two to three males per million. Fewer than 10 cases with definite gene mutations have been reported in Chinese mainland and no rapid diagnosis method has been established. PROCEDURE: We determined the clinical and molecular characteristics of five patients with XLP1. The SH2D1A gene were amplified by PCR and sequenced, the SAP expression was analyzed by flow cytometry. RESULTS: Two patients had novel SH2D1A mutations and three had mutations that have been previously reported. Three patients presented with fulminant infectious mononucleosis or hemophagocytic lymphohistiocytosis and one presented with lymphoma. Null or decreased SAP expression on PBMCs was noted. The remaining patient presented with unique, recurrent, nonfulminant infectious mononucleosis and bimodal intracellular SAP protein expression. CONCLUSIONS: The overall molecular characteristics and clinical phenotypes of Chinese patients with XLP1 matched previous reports. The unique bimodal intracellular SAP protein expression indicated the presence of some residual SAP-positive T cells that are able to respond to persistent Epstein-Barr virus infection and could explain the relatively mild clinical phenotype of this patient.

Clinical, molecular, and T cell subset analyses in a small cohort of Chinese patients with hyper-IgM syndrome type 1.

Type 1 hyper-IgM syndrome (HIGM1) is a rare primary immunodeficiency disease caused by mutations in the CD40L gene. Patients often present with recurrent infections and autoimmune manifestations. We investigated the clinical and molecular characteristics of HIGM1 in thirteen patients from the Chinese mainland and examined the proportion of CD4(+)CD25(+)FoxP3(+)Treg, Th17, and Th1 cells in the peripheral blood. We identified ten distinct CD40L mutations in eleven patients: one missense mutation, one nonsense mutation, one insertion mutation (in frame), and seven deletions. Six of these mutations were novel. We observed the percentage of Tregs in the peripheral blood of HIGM1 patients decreased markedly compared with that in healthy controls, but no statistically significant differences was found in the percentages of Th17 and Th1. The identified mutations reflect the heterogeneity of the CD40L gene in HIGM1. Precise genetic diagnosis of HIGM1 will enable appropriate therapeutic interventions, reliable detection of carriers, and genetic counseling. Skewed Treg, Th17/Treg, and Th1/Treg profiles may be associated with immune responses to autoimmunity or infection, which requires replication in larger studies.

[Expression and significance of interleukin-6, interferon-inducible protein-10 and interleukin-17 in serum and synovial fluid of patients with juvenile idiopathic arthritis].

OBJECTIVE: To detect the disparity of three cytokines interleukin-6 (IL-6), interferon-inducible protein 10 (IP-10) and interleukin-17 (IL-17) in peripheral blood (PB) and synovial fluid (SF) of patients with juvenile idiopathic arthritis (JIA). METHOD: Serum concentrations of the three cytokines were measured in 27 patients with 13 systemic-onset JIA (sJIA), 14 polyarticular JIA (pJIA) and 28 healthy controls using enzyme-linked immunosorbent assay (ELISA). Nineteen patients with no marked arthritis symptom or only temporary arthralgia were enrolled in probable sJIA group. SF from 18 patients with 7 sJIA, 11 pJIA were examined for cytokine levels. RESULT: (1) The statistically significant difference in serum IL-6 was detected between sJIA and healthy control group [28.0(4.2-59.2) ng/L vs. 12.3 (2.1-13.8) ng/L, P < 0.05], but no significant difference between probable sJIA and healthy control group [11.8(7.7-39.2) ng/L vs. 12.3 (2.1-13.8) ng/L, P > 0.05] was found. There were statistically significant differences between sJIA group and healthy control group in serum concentrations of IL-17 [14.0(9.8-34.3) ng/L vs. 9.8 (7.9-16.2) ng/L, P < 0.05], yet compared to healthy control group, no significant difference in concentration level of IL-17 was found in pJIA Group [14.2(9.9-16.9) ng/L vs. 9.8(7.9-16.2) ng/L, P > 0.05].(2) In sJIA and pJIA SF, the median IP-10 level was significantly higher compared to respective PB levels [619.7 (160.9, 873.1) ng/L vs. 64.8 (27.4-111.9) ng/L;660.9 (401.9, 1349.8) ng/L vs. 97.4 (41.9-222.1) ng/L, P < 0.01, respectively], but there was only significant difference in IL-17 between pJIA SF and PB [22.9 (17.1, 45.8) ng/L vs. 14.2 (9.9-16.9) ng/L, P < 0.01]. CONCLUSION: IL-6 may play more important role in the pathogenesis of sJIA. Moreover, IL-6 may be the biomarker associated with arthritis in early JIA stage. Both autoinflammation and autoimmune response may be involved in the pathogenesis of sJIA. IL-17 enrichment may only occur in local joint, the levels of IL-17 in PB may not be significantly increased. The prominent expression gradient between SF and PB of IP-10 maybe the basis of performing chemotaxis and further causing joint damage.

Distribution, clinical features and molecular analysis of primary immunodeficiency diseases in Chinese children: a single-center study from 2005 to 2011.

METHODS: Two hundred three children with genetically proven primary immunodeficiency diseases (PIDs) from 197 unrelated families were enrolled from January 2005 to December 2011. RESULTS: On the basis of criteria developed by the International Union of Immunological Societies, 79 patients were diagnosed as "other well-defined immunodeficiency syndromes" (38.9%), 62 (30.6%) with "predominant antibody deficiencies," 26 (12.8%) with "congenital defects of phagocyte," 25 (12.3%) with "T- and B-cell immunodeficiency" and 11 (5.4%) with "diseases of immune dysregulation." The median time to the diagnosis was 27.9 months and the patients had a wide range of clinical presentations. In addition, a total of 23 pathogenic genes were identified and 213 mutations were detected, including 42 novel mutations. CONCLUSIONS: With the increase in the awareness of PIDs and diagnostic competence, more PID patients will be diagnosed and we will be able to more accurately identify the frequency and the distribution of PIDs in the most populous country in the world.

[Screening for cytotoxic defects with flow cytometric detection of CD107α on natural killer cells and cytotoxic lymphocyte cells].

OBJECTIVE: To establish a novel flow cytometry-based assay for measuring the expression of lysosomal-associated membrane protein 1 (LAMP-1, CD107α) on the cell surface of natural killer (NK) cells and cytotoxic T lymphocyte (CTL) and evaluate the screening value of this assay for cytotoxic defects-related diseases such as familial hemophagocytic lymphopro-liferative (FHL) syndrome. METHOD: Three suspected Chediak-Higashi Syndrome (CHS) patients, three suspected FHL patients and 10 healthy children were enrolled in the study from October 2010 to June 2011. Their PBMCs were separated and activated overnight with IL-2. After the granule release of NK cells activated by phytohemagglutinin (PHA) and CD8+T cells by anti-CD3, the CD107α expression were analyzed by flow cytometry. The peripheral blood DNA and RNA of the patients were extracted to analyze the pathogenic genes via DNA-PCR/RT-PCR and direct sequencing. RESULT: The CD107α expression on CTL in the ten healthy children significantly increased after activation by anti-CD3 [(0.18 ± 0.07)% vs. (4.47 ± 2.36)%, P < 0.05] and NK cells after activation by PHA [(0.27 ± 0.07)% vs. (5.80 ± 2.83)%, P < 0.05]. The frequency of CD107α-expression NK cells in three suspected CHS after activation was significantly elevated when compared with the healthy control [0.5%, 0.6% vs. (5.80 ± 2.83)%] except patient 2. After the anti-CD3 activation, the frequency of CD107α expression on CTL cells also showed no significant difference [0.3%, 0.9%, 0.2% vs. (4.47 ± 2.36)%] in three patients. All of their mean fluorescence intensity (MFI) showed the same trend. Patient 1 and 3 were identified to have LYST mutations (Patient 1: c.5411-5414 del TTTC, L1741fsX1758 and c.7975 C > T, R2596X; Patient 3: c.4863G > A, R1563H and c.5392-5393delAA, E1739fsX1756). There was no mutation identified in the LYST gene for patient 2. CD107α expression of NK cells and CTL in the suspected FHL patients and in mirror of these findings, no underlying gene variation of PRF, MUNC13-4 and STX11 were identified. CONCLUSION: We developed a method to quantitatively assess cytotoxicity of the NK cells and CTL by measuring the expression of CD107α on the cell membrane, which appeared to be an effective and rapid screening test for cytotoxic defects-related diseases such as FHL and other HLH secondary to primary immunodeficiency.

[Genetic and proteinic analysis of a Chinese boy with X-linked lymphoproliferative disease and his maternal relatives].

OBJECTIVE: X-linked lymphoproliferative disease (XLP), a genetic disorder characterized by immunodeficiency to Epstein-Barr virus (EBV) infection, has been linked to mutations in the SH2D1A gene. XLP patient displays EBV associated fulminant infectious mononucleosis or hemophagocytic lymphohistocytosis, hypogammaglobulinemia or malignant lymphoma. Here we report the clinical features, gene mutation and SAP expression on PBMCs of a Chinese patient with XLP and potential carriers. METHOD: A 6 years old male patient and his maternal relatives were enrolled in this study. The patient was found to have with a renal Burkitt lymphoma on the right waist at 5 years of age by accident. His elder brother and a maternally related cousin both died of multiple systemic organ dysfunction syndrome (MODS) due to fulminant infectious mononucleosis (FIM) at the age of one year. The patient and his maternal relatives were subjected to detection of SAP expression on the PBMCs by flow cytometry and gene mutation analysis of SH2D1A by using PCR based on genomic DNA. RESULT: The patient exhibited 536.9 copy/ml level of circulating EBV-DNA during remission. Sequence analysis showed that the patient harbored a nonsense mutation in exon 2 (C462T), resulting in a premature stop codon (Arg55X). His mother and some of the maternal relatives were proved to be carriers of this mutation. SAP expression from the patient was significantly reduced as compared to normal individual and the carriers. CONCLUSION: We identified a Chinese XLP case genetically. Assessment of SAP expression on PBMCs by flow cytometry seemed to be an effective rapid diagnostic method for this disease. Absence of EBV infection does not diminish the possibility of XLP.

Clinical characteristics and molecular analysis of three Chinese children with Omenn syndrome.

Omenn syndrome (OS) is a rare autosomal recessive genetic disorder and presents symptoms of severe combined immunodeficiency characterized by erythrodermia, eosinophilia, hepatosplenomegaly, lymphadenopathy, and elevated serum IgE levels. OS has been found to be caused by mutations in RAG1 or RAG2 gene that result in partial V(D)J recombination activity. No study on OS has been reported in Chinese children so far. In this study, the genotype and phenotypes of three infants with OS from three unrelated Chinese families were investigated. All the three children had most of the characteristics of OS except normal serum IgE level. Compound heterozygosity mutations in RAG1 gene (1983 G>A; 2444 C>T and 2219 C>T; 3127 C>G) were identified in two cases, and a homozygous deletion mutation with a premature stop codon was found at residue 2302 of RAG1 gene (2302delT, I729X) in the remaining case, including three novel mutations (2302delT, I729X; 2219 C>T, R699W; and 3127 C>G, Y1001X). Spectratyping analysis of T-cell receptor ß-chain variable region (TCRVß) gene rearrangement was performed in case 1 and case 2. All the 25 TCRVß subfamilies presented monoclonal or oligoclonal peaks in case 1 and 11 TCRVß subfamilies were very weak or even absent in case 2. This was the first report about OS in Chinese children. Molecular genetic testing represents an important tool for early confirmed diagnosis and may allow accurate carrier detection and prenatal diagnosis.

[A compound heterozygosity mutation in the interleukin-7 receptor-alpha gene resulted in severe combined immunodeficiency in a Chinese patient].

OBJECTIVE: Mutation in the interleukin-7 receptor-alpha (IL-7R alpha) chain causes a rare type of severe combined immunodeficiency (SCID) with presence of NK cells in the peripheral blood. Here we report the molecular and clinical characterization of a compound heterozygosity mutation in the interleukin-7 receptor-alpha gene that resulted in SCID in a patient firstly from China. METHOD: A 5 month-old male patient and his parents were enrolled in this study. Since 15 days of age, the patient had had recurrent fever, persistent cough and diarrhea. He was in poor general condition with pyorrhea and ulceration of the BCG scar. His brother died of severe infection at 4 months of age. He was initially diagnosed as SCID according to clinical manifestation and immunological analysis. A panel of SCID candidate genes including IL-2RG, RAG1/RAG2 and IL-7R alpha of patient and his parents were amplified by polymerase chain reaction (PCR) from genomic DNA. Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify the IL-7R alpha transcripts. Sequencing was performed directly on the PCR products forward and reversely. RESULT: The serum immunoglobulin (Ig) profile was IgG 6867 mg/L (normal range, 3050 - 8870 mg/L); IgM 206 mg/L and IgA 249 mg/L, IgE 2.3 IU/ml (normal range < 150 IU/ml). The patient was treated with IVIG previously. There were no T-cells but increased percentage of B-cells (58%) and NK cells (42%) in the peripheral blood was found. Needle biopsies from enlarged axillary lymph node was identified positive for Mycobacterium bovis under microscope and by culture. The patient had a compound heterozygosity mutation in the IL-7R alpha gene:on one allele, there was a splice-junction mutation in intron 4 (intron 4(+1)G > A), for which his father was a carrier; whereas on the other allele, a nonsense mutation at position 638 in exon 5 with a premature stop codon (638 C > T, R206X) was identified, for which his mother was a carrier. The splice-junction mutation in intron 4 of IL-7R alpha was firstly reported. The IL-7R alpha mRNA expression of the patient was remarkably reduced whereas the parents had relatively normal IL-7R alpha mRNA expression. IL-7R alpha cDNA of the patient was amplified by nested PCR. The PCR products were purified, cloned with a TA Cloning Kit and sequenced directly. A 64 bp deletion was found in exon 4 of IL-7R alpha. No mutation was found in IL-2RG and RAG1/RAG2 of the patient and his parents. CONCLUSION: This is the first case with a compound heterozygosity mutation in the IL-7R receptor alpha gene and T-B+NK+ phenotype from China. Intron 4(+1)G > A was a novel mutation.

[A novel missense mutation of FOXP3 causes immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome in a Chinese child].

OBJECTIVE: To investigate variation of FOXP3 and it's expression in male children presented with severe and early-onset enteropathy, rash with or without insulin-dependent diabetes mellitus (IDDM). METHODS: Four male children presented with severe and early-onset enteropathy, rash, with or without IDDM were subjected to detection of FOXP3 expression on the PBMC by flow cytometry and FOXP3 gene analysis. The maternal gene analysis was subsequently performed once the variant FOXP3 gene was found. All 11 exons and splice sites within FOXP3 gene were amplified by polymerase chain reaction (PCR) from genomic DNA. Reverse transcription polymerase chain reaction was used to amplify the FOXP3 transcripts. Sequence analysis was performed directly on the bulk PCR products forwardly and reversely. The candidate mutation site was compared with that of 100 healthy controls to exclude polymorphism. Flow cytometry was used to determine FOXP3 expression on CD4+CD25+ T cells and the frequency of Tregs in CD4+ T cells. RESULTS: One of the 4 patients showed a G13128A genetic variation in exon 11, which resulted in a Met370Ile substitution. No sequence variations were found at the same site in any of 100 healthy controls, indicating that the Met370Ile substitution is not a polymorphism but a novel missense mutation. The patient's mother was identified as a carrier for this mutation. There was no reduced frequency of Tregs in the peripheral blood of the patient and FOXP3 protein expression is normal as compared with controls. CONCLUSION: A novel missense mutation of FOXP3 which causes IPEX phenotype was identified in a Chinese child according to immunologic screening and gene sequencing. Infants with early-onset IDDM and persistent diarrhea should be suspected as IPEX, FOXP3 gene analysis will be a reliable diagnostic approach to IPEX.