Objective: To analyze the clinical phenotype and screen the genetic mutations of hereditary deafness in three deaf families to clarify their molecular biology etiology. Methods: From January 2019 to January 2020, three deaf children and family members were collected for medical history, physical examination, audiology evaluation, electrocardiogram and cardiac color Doppler ultrasound, temporal bone CT examination, and peripheral blood DNA was obtained for high-throughput sequencing of deafness genes. Sanger sequencing was performed to verify the variant sites among family members. The pathogenicity of the variants was evaluated according to the American College of Medical Genetics and Genomics. Results: The probands in the three families had deafness phenotypes. In family 1, proband had multiple lentigines, special facial features, growth retardation, pectus carinatum, abnormal skin elasticity, cryptorchidism and other manifestations. In family 2, proband had special facial features, growth retardation and abnormal heart, and the proband in family 3 had growth retardation and abnormal electrocardiogram. Genetic testing of three families detected three heterozygous mutations in the PTPN11 gene: c.1391G>C (p.Gly464Ala), c.1510A>G (p.Met504Val), c.1502G>A (p.Arg501Lys). All three sites were missense mutations, and the mutation sites were highly conserved among multiple homologous species. Based on clinical manifestations and genetic test results, proband 1 was diagnosed with multiple lentigines Noonan syndrome, and probands 2 and 3 were diagnosed with Noonan syndrome. Conclusion: Missense mutations in the PTPN11 gene may be the cause of the disease in the three deaf families. This study enriches the clinical phenotype and mutation spectrum of the PTPN11 gene in the Chinese population.
Deafness/genetics* ; Genetic Testing ; Hearing Loss/genetics* ; Humans ; Male ; Mutation ; Phenotype ; Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics*

OBJECTIVE: To analyze the gene mutations of children with juvenile myelomonocytic leukemia (JMML) and their correlation with clinical characteristics. METHODS: The genetic mutation results and clinical data of 19 children with JMML in Fujian from January 2015 to December 2018 were collected and analyzed retrospectively. According to the results of gene mutation, they were divided into PTPN11 gene mutation group and non-PTPN11 gene mutation group, and the clinical characteristics and prognosis of children with JMML between two groups were compared. RESULTS: Among the 19 children with JMML, 14 cases were male and 5 cases were female, and male/female ratio was 2.8∶1. The median age at diagnosis was 13(3-48) months, and 14 cases (73.68%) were less than 2 years old. Abdominal distension and pyrexia were the common initial symptoms, and all the children with JMML had splenomegaly. The median white blood cell count was 39.82(4.53-103.4)×10 CONCLUSION JMML is more common in male infancy and toddlerhood, and the main gene mutation types are PTPN11 and Ras mutations. Because the JMML children with PTPN11 mutations show particularly rapid disease progression, if there is no timely intervention, most children die in a short period of time. Therefore, early HSCT may improve the prognosis of the children with JMML.
Child ; Female ; Hematopoietic Stem Cell Transplantation ; Humans ; Infant ; Leukemia, Myelomonocytic, Juvenile/genetics* ; Male ; Mutation ; Prognosis ; Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics* ; Retrospective Studies

Two new 2-carboxymethyl-3-hexyl-maleic anhydride derivatives, arthrianhydride A (1) and B (2), along with three known compounds 3-5, were isolated from the fermentation broth of a grasshopper-associated fungus Arthrinium sp. NF2410. The structures of new compounds 1 and 2 were determined based on the analysis of the HR-ESI-MS and NMR spectroscopic data. Furthermore, compounds 1 and 2 were evaluated on inhibitory activity against the enzyme SHP2 and both of them showed moderate inhibitory activity against SHP2.
Anhydrides/pharmacology* ; Animals ; Biological Products/pharmacology* ; Enzyme Inhibitors/pharmacology* ; Fungi/chemistry* ; Grasshoppers/microbiology* ; Molecular Structure ; Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors* ; Secondary Metabolism

The transition from spermatogonia to spermatocytes and the initiation of meiosis are key steps in spermatogenesis and are precisely regulated by a plethora of proteins. However, the underlying molecular mechanism remains largely unknown. Here, we report that Src homology domain tyrosine phosphatase 2 (Shp2; encoded by the protein tyrosine phosphatase, nonreceptor type 11 [Ptpn11] gene) is abundant in spermatogonia but markedly decreases in meiotic spermatocytes. Conditional knockout of Shp2 in spermatogonia in mice using stimulated by retinoic acid gene 8 (Stra8)-cre enhanced spermatogonial differentiation and disturbed the meiotic process. Depletion of Shp2 in spermatogonia caused many meiotic spermatocytes to die; moreover, the surviving spermatocytes reached the leptotene stage early at postnatal day 9 (PN9) and the pachytene stage at PN11-13. In preleptotene spermatocytes, Shp2 deletion disrupted the expression of meiotic genes, such as disrupted meiotic cDNA 1 (Dmc1), DNA repair recombinase rad51 (Rad51), and structural maintenance of chromosome 3 (Smc3), and these deficiencies interrupted spermatocyte meiosis. In GC-1 cells cultured in vitro, Shp2 knockdown suppressed the retinoic acid (RA)-induced phosphorylation of extracellular-regulated protein kinase (Erk) and protein kinase B (Akt/PKB) and the expression of target genes such as synaptonemal complex protein 3 (Sycp3) and Dmc1. Together, these data suggest that Shp2 plays a crucial role in spermatogenesis by governing the transition from spermatogonia to spermatocytes and by mediating meiotic progression through regulating gene transcription, thus providing a potential treatment target for male infertility.
Animals ; Cell Cycle Proteins/genetics* ; Cell Line ; Cell Survival ; Chondroitin Sulfate Proteoglycans/genetics* ; Chromosomal Proteins, Non-Histone/genetics* ; Gene Expression Regulation ; Gene Knockdown Techniques ; Infertility, Male ; Male ; Meiosis/genetics* ; Mice ; Mice, Knockout ; Mice, Transgenic ; Phosphate-Binding Proteins/genetics* ; Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics* ; Rad51 Recombinase/genetics* ; Real-Time Polymerase Chain Reaction ; Spermatocytes/metabolism* ; Spermatogenesis/genetics* ; Spermatogonia/metabolism*

Male ; Meiosis ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; Spermatocytes ; Spermatogenesis ; Spermatogonia ; Tyrosine

OBJECTIVETo detect the expression of PTPN11 gene in acute myeloid leukemia (AML) cell line and to explore the effects of PTPN11 over expressing on proliferation and apotosis of AML cell lines.

METHODSThe expression of PTPN11 in AML cell lines（HEL,U937, K562, KG-1, HL -60） was detected by RT-PCR, Q-PCR and Western blot. The PTPN11 gene was amplified by RT-PCR. PTPN11 DNA fragement and the lentiviral vector PCDH-CD513B were digested by BamHI and EcoRI, and then ligated by T4 DNA ligase. Recombinant lentivirus was generated by co-transfection of three-plasmids into 293FT cells using lipofectamine 2000. Then Q-PCR and Western blot were used to detect the expression of PTPN11 in the lentivirus infected HEL and U937 cells. The CCK-8 and Annexin V/7-AAD assays were performed to evaluate effects of PTPN11 on proliferation, apoptosis of HEL and U937 cells.

RESULTSAll 5 AML cell lines expressed the PTPN11 gene, restriction analysis and gene sequencing confirmed that recombinant lentiviral vector was successfully constructed. After transfection of cells with the lentivirus, the recombinant plasmid could stably up-regulate the expression of PTPN11. Analysis of the proliferation and apoptosis of transfected AML cells indicated that as compared with the control group, the OD values of over-expression group were significantly higher and the apoptotic rates were significantly lower (P<0.05).

CONCLUSIONPTPN11 is expressed in all the 5 AML cell lines. The lentiviral expression vector carrying human PTPN11 and the engineered HEL and U937 cell lines stably up-regulating PTPN11 gene expression are successfully obtained. Over-expression of PTPN11 promotes the proliferation of AML cell lines and inhibit then apoptosis.

Apoptosis ; Cell Line, Tumor ; Gene Expression ; Genetic Vectors ; Humans ; Lentivirus ; Leukemia, Myeloid, Acute ; Plasmids ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; Transfection

Mutation of leucine-rich repeat kinase 2 (LRRK2) causes an autosomal dominant and late-onset familial Parkinson's disease (PD). Recently, we reported that LRRK2 directly binds to and phosphorylates the threonine 474 (T474)-containing Thr-X-Arg(Lys) (TXR) motif of focal adhesion kinase (FAK), thereby inhibiting the phosphorylation of FAK at tyrosine (Y) 397 residue (pY397-FAK), which is a marker of its activation. Mechanistically, however, it remained unclear how T474-FAK phosphorylation suppressed FAK activation. Here, we report that T474-FAK phosphorylation could inhibit FAK activation via at least two different mechanisms. First, T474 phosphorylation appears to induce a conformational change of FAK, enabling its N-terminal FERM domain to autoinhibit Y397 phosphorylation. This is supported by the observation that the levels of pY397-FAK were increased by deletion of the FERM domain and/or mutation of the FERM domain to prevent its interaction with the kinase domain of FAK. Second, pT474-FAK appears to recruit SHP-2, which is a phosphatase responsible for dephosphorylating pY397-FAK. We found that mutation of T474 into glutamate (T474E-FAK) to mimic phosphorylation induced more strong interaction with SHP-2 than WT-FAK, and that pharmacological inhibition of SHP-2 with NSC-87877 rescued the level of pY397 in HEK293T cells. These results collectively show that LRRK2 suppresses FAK activation through diverse mechanisms that include the promotion of autoinhibition and/or the recruitment of phosphatases, such as SHP-2.
Focal Adhesion Protein-Tyrosine Kinases ; Glutamic Acid ; Parkinson Disease ; Phosphoric Monoester Hydrolases ; Phosphorylation ; Phosphotransferases ; Protein Tyrosine Phosphatase, Non-Receptor Type 11* ; Threonine ; Tyrosine*

BACKGROUNDSrc homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) is a kind of intracellular protein tyrosine phosphatase. Studies have revealed its roles in various disease, however, whether SHP-2 involves in renal fibrosis remains unclear. The aim of this study was to explore the roles of myeloid cells SHP-2 in renal interstitial fibrosis.

METHODSMyeloid cells SHP-2 gene was conditionally knocked-out (CKO) in mice using loxP-Cre system, and renal interstitial fibrosis was induced by unilateral ureter obstruction (UUO). The total collagen deposition in the renal interstitium was assessed using picrosirius red stain. F4/80 immunostaing was used to evaluate macrophage infiltration in renal tubular interstitium. Quantitative real-time polymerase chain reaction and enzyme linked immunosorbent assay were used to analyze the production of cytokines in the kidney. Transferase-mediated dUTP nick-end labeling stain was used to assess the apoptotic renal tubular epithelial cells.

RESULTSSrc homology 2 domain-containing protein tyrosine phosphatase-2 gene CKO in myeloid cells significantly reduced collagen deposition in the renal interstitium after UUO. Macrophage infiltration was evidently decreased in renal tubular interstitium of SHP-2 CKO mice. Meanwhile, the production of pro-inflammatory cytokines was significantly suppressed in SHP-2 CKO mice. However, no significant difference was observed in the number of apoptotic renal tubular epithelial cells between wild-type and SHP-2 CKO mice.

CONCLUSIONSOur observations suggested that SHP-2 in myeloid cells plays a pivotal role in the pathogenesis of renal fibrosis, and that silencing of SHP-2 gene in myeloid cells may protect renal from inflammatory damage and prevent renal fibrosis after renal injury.

Animals ; Enzyme-Linked Immunosorbent Assay ; Female ; Fibrosis ; enzymology ; pathology ; Immunohistochemistry ; Kidney Diseases ; enzymology ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Myeloid Cells ; metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; genetics ; metabolism ; Ureteral Obstruction ; enzymology ; pathology

OBJECTIVETo study gene mutations and clinical features in children with juvenile myelomonocytic leukemia (JMML).

METHODSThe clinical data of 14 children who were diagnosed with JMML and were examined for the detection of common gene mutations were retrospectively analyzed.

RESULTSEleven (79%) out of 14 cases were male, and 3 (21%) were female. The median age at diagnosis was 2.0 years (age range: 0.6-6.0 years). Among 14 cases, there were 4 cases (29%) with PTPN11 mutation, 3 cases (21%) with N-RAS mutation, 1 case (7%) with PTPN11 mutation and K-RAS mutation, and 6 cases (43%) without any mutation. All four cases in the PTPN11 mutation group were male, and their median age was 2.5 years; interval from onset to diagnosis was 1.0 month; the white blood cell (WBC) count and absolute monocytes in peripheral blood were significantly higher, while the platelet (PLT) count was lower, as compared with the other three groups; they were followed up, and 3 cases died and 1 case had a progressive disease. In the N-RAS mutation group, there were two male cases and one female case, and their median age was 2.0 years; interval from onset to diagnosis was 13.7 months; after follow-up, 2 cases died and 1 case did not have an obviously progressive disease.

CONCLUSIONSPTPN11 mutation is the most common mutation in JMML. The cases with PTPN11 mutation often have higher WBC count and absolute monocytes in peripheral blood, a lower PLT count, and a rapid disease progression, and their clinical outcomes are poor. The cases with N-RAS mutation have a slow disease progression. The clinical characteristics of the patients with compound mutations are not sure because of the small number of cases, and further clinical observation is indispensable.

Child ; Child, Preschool ; Female ; Genes, ras ; Humans ; Infant ; Leukemia, Myelomonocytic, Juvenile ; blood ; genetics ; Male ; Mutation ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; genetics ; Retrospective Studies

OBJECTIVETo investigate the effects of Bortezomib on proliferation, apoptosis and SHP-2 gene expression of lymphoma Jurkat cells and Raji cells.

METHODSMethylthiazoly tetrazolium assay (MTT) was used to observe the proliferation of Jurkat cells and Raji cells treated with bortezomib in different doses. Cell apoptosis was detected by morphological examination and flow cytometry. The level of SHP-2 mRNA expression before and after the treatment with bortezomib was measured by RT-PCR.

RESULTSBortezomib could inhibit the proliferation of Jurkat and Raji cells and induce their apoptosis with time-and dose-dependent manner. After treatment with 5-100 nmol/L bortezomib, the expression of SHP-2 in Jurkat cells and Raji cells was upregulated.

CONCLUSIONBortezomib can inhibit the proliferation and induc the apoptosis of Jurkat and Raji cells obviously, upregulate the expression of SHP-2 mRNA, suggesting that the SHP-2 may participate in regulation of bortezomib induced apoptosis of Jurkat cells and Raji cells.

Apoptosis ; Bortezomib ; Cell Line, Tumor ; Cell Proliferation ; Flow Cytometry ; Gene Expression Regulation, Leukemic ; Humans ; Lymphoma ; genetics ; pathology ; Protein Tyrosine Phosphatase, Non-Receptor Type 11