Targeted high-throughput sequencing technique for the molecular diagnosis of primary immunodeficiency disorders.

The aim of this study was to investigate the usefulness of targeted high-throughput sequencing (HTS) for the molecular diagnosis of primary immunodeficiency diseases (PID).A total of 56 clinically diagnosed or suspected PID patients were divided into 4 groups according to the International Union of Immunological Societies Expert Committee for Primary Immunodeficiency 2015 and their chief clinical presentations. Patients and their biological family members were examined by targeted HTS, which sequenced the exons and ±10 bp flanking introns of 171 PID-related genes panel. All significant variants were confirmed by PCR-Sanger sequencing. Pathogenicity of the variants was evaluated by using bioinformatics.A total of 117 variants in 73 genes were found in 56 patients. Accurate molecular diagnosis of PID was made in 13 (23.2%) patients, and 12 novel mutations were detected in these patients. Twenty-seven patients carried heterozygous variants that are probably pathogenic in ≥2 genes; 16 patients had only 1 missense variant, or had several variants but not >1 variant was deleterious as evaluated by bioinformatics. The meaning of the targeted HTS results of these patients remains to be studied.Targeted HTS can make a precise molecular diagnosis of PID and detect more novel pathogenic mutations. More and more variations with ambiguous significance are discovered and explanation of these variations is a challenge to the clinicians.

[Gene Mutation and Acute Leukemia Transformation of Severe Congenital Neutropenia- Review].

Severe congenital neutropenia (SCN) is a rare disease of bone marrow failure. Absolute value of peripheral blood neutrophil of SCN significantly reduced. SCN has a high risk of transformation to myelodysplastic syndromes (MDS) / acute myeloid leukemia (AML). At present, there are 14 abnormal genes related to SCN, ELANE is the most common pathogenic gene, the main therapy of SCN is the application of granulocyte colony stimulating factor (G-CSF). CSF3R gene mutation often occurs in the treatment process, and can lead to acute myeloid leukemia. Further research on SCN/AML transformation mechanism is helpful to the diagnosis and treatment of this disease. This review focuses on the genetics and phenotypic polymorphysm in SCN patients, the therapeutic effect and risk of G-CSF for SCN patients, the effect of CSF3R matation on signal transduction of G-CSF, CSF3R mutation is important factor for SCN tranformation to acute myeloid leukemia, exploring the mechanism of SCN/AML transformation contributes to diagnosis and therapy for patients and so on.

[Study on differentiation of mesenchymal stem cells derived from human umbilical cord blood into insulin secreting cells].

OBJECTIVE: To investigate the differential potential of mesenchymal stem cells (MSCs) derived from human umbilical cord blood (hUCB) into insulin-secreting cells and its inducing condition. METHODS: UCB nucleated cells (NCs) were isolated and cultured in Mesencult media. The obtained UCB MSC were purified by adherence method and expanded. Then they were induced with epidermal growth factor (EGF), B-mercaptoethanol and high concentration of glucose. The induced cells were identified by RT-PCR. Intracellular insulin was examined by immunocytochemistry. The quantity of insulin secretion and glucose-simulated insulin release were examined by chemiluminescence immunoassay. The induced cells were also transplanted into renal subcapsular space of STZ-induced hyperglycemic mice to observe the in vivo lowering effect on hyperglycemia. RESULTS: The induced cells morphologically became round and were gathering into a mass. The expression of some genes related to pancreatic islet was found by RT-PCR. Chemiluminescence immunoassay showed insulin positivity and the cells secreted a low concentration of insulin [(0.37 +/- 0.06) mU/L]. The induced cells responded to high glucose challenge with a stimulation index of 1.76. After those cells grafted into renal sub-capsule there was an in vivo lowering effect on blood glucose level on STZ hyperglycemic mice. CONCLUSION: MSCs from UCB can differentiated into insulin secreting cells.

[Expression of lung resistance protein and multidrug resistance protein genes in bone marrow cells of acute leukemia patients and its clinical significance].

To study the expression of lung resistance protein (LRP) and multidrug resistance protein (MRP) genes in bone marrow cells in patients with acute leukemia and its clinical significance, expression of LRP and MRP mRNA in bone marrow cells from 47 cases of acute leukemia, including 10 refractory or relapsed cases, and 7 normal individuals were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The result s showed that expression of LRP gene was negative in normal individuals. LRP mRNA level in newly treated cases of acute myelocytic leukemia and refractory or relapsed cases was significantly higher than that in normal individuals, increased LRP mRNA level has correlation with lower sensitivity to initial chemotherapy and was associated with reduced overall survival rate. Complete remission (CR) rate in LRP positive patients was lower than that in negative cases. The level of LRP expression was correlated with that of MRP mRNA. In conclusion, the expression of LRP mRNA can predict the treatment outcome and prognosis for acute myelocytic leukemia, prognosis was even worse in LRP and MRP linked expression cases, therefore, LRP was an important resistant factor, determination of LRP and MRP expression can help us to evaluate the prognosis and choose chemotherapy program.

[Expression of nm23-H(1) mRNA in Bone Marrow Cells from Patients with Myelodysplastic Syndrome and Its Clinical Implication]

The purpose of this investigation was to explore the expression of nm23-H(1) gene in patients with myelodysplastic syndrome (MDS) and evaluate the relationship between nm23-H(1) expression and therapeutic outcomes. Semi-quantitative RT-PCR was used to detect the expression of nm23-H(1) mRNA in marrow mononuclear cells from 28 MDS patients and 15 normal subjects. nm23-H(1)/GAPDH ratio >/= 0.5 was believed to a positive case. The expression of nm23-H(1) was positive in 24 of 28 MDS patients, and the average level was 0.89 +/- 0.56. nm23-H(1) mRNA was negative in normal controls. The overexpression of nm23-H(1) mRNA in MDS patients could predict outcome of treatment and prognosis for MDR patients.