Objective: To investigate the clinical presentation and genetic characteristics of malignant infantile osteopetrosis. Methods: This was a retrospective case study. Thirty-seven children with malignant infantile osteopetrosis admitted into Beijing Children's Hospital from January 2013 to September 2022 were enrolled in this study. According to the gene mutations, the patients were divided into the CLCN7 group and the TCIRG1 group. Clinical characteristics, laboratory tests, and prognosis were compared between two groups. Wilcoxon test or Fisher exact test were used in inter-group comparison. The survival rate was estimated with the Kaplan-Meier method and the Log-Rank test was used to compare the difference in survival between groups. Results: Among the 37 cases, there were 22 males and 15 females. The age of diagnosis was 0.5 (0.2, 1.0) year. There were 13 patients (35%) and 24 patients (65%) with mutations in CLCN7 and TCIRGI gene respectively. Patients in the CLCN7 group had an older age of diagnosis than those in the TCIRGI group (1.2 (0.4, 3.6) vs. 0.4 (0.2, 0.6) years, Z=-2.60, P=0.008). The levels of serum phosphorus (1.7 (1.3, 1.8) vs. 1.1 (0.8, 1.6) mmol/L, Z=-2.59, P=0.010), creatine kinase isoenzyme (CK-MB) (457 (143, 610) vs. 56 (37, 82) U/L, Z=-3.38, P=0.001) and the level of neutrophils (14.0 (9.9, 18.1) vs. 9.2 (6.7, 11.1) ×10⁹/L, Z=-2.07, P=0.039) at diagnosis were higher in the CLCN7 group than that in the TCIRG1 group. However, the level of D-dimer in the CLCN7 group was lower than that in the TCIRGI group (2.7 (1.0, 3.1) vs. 6.3 (2.5, 9.7) μg/L, Z=2.83, P=0.005). After hematopoietic stem cell transplantation, there was no significant difference in 5-year overall survival rate between the two groups (92.3%±7.4% vs. 83.3%±7.6%, χ²=0.56, P=0.456). Conclusions: TCIRGI gene mutations are more common in children with osteopetrosis. Children with TCIRGI gene mutations have younger age, lower levels of phosphorus, CK-MB, and neutrophils and higher level of D-dimer at the onset. After hematopoietic stem cell transplantation, patients with CLCN7 or TCIRGI gene mutations have similar prognosis.
Child ; Male ; Female ; Humans ; Osteopetrosis/therapy* ; Retrospective Studies ; Prognosis ; Genes, Recessive ; Phosphorus ; Chloride Channels/genetics* ; Vacuolar Proton-Translocating ATPases/genetics*

Vacuolar-type ATPase (V-ATPase), located in the membrane and organelle membrane, is one of important H⁺-transporting proteins. It keeps the proton balance by transporting H⁺ into vacuole, vesicle, or extracellular using the energy from ATP hydrolysis. The subunit B of the vacuolar-type ATPase (BmV-ATPase B) contains the ATP catalytic site, and plays an important role in this process. To study the function of V-ATPase B in Bombyx mori (BmV-ATPase B), we cloned its coding gene from the midgut of the 5th instar silkworm larvae. Then we constructed prokaryotic expression vector and produced the recombinant protein in E. coli. The recombinant protein was identified as BmV-ATPase B by mass spectrometry and purified using Ni-NTA affinity chromatography. This purified protein was used to immunize rabbit to generate polyclonal antibodies of BmV-ATPase B. Finally, the expression patterns of BmV-ATPase B in the silk gland were analyzed by western blotting and immunofluorescence. The full length CDS sequence of BmV-ATPase B was 1 473 bp. BmV-ATPase B was 55 kDa with a PI of 5.3. We analyzed the expression patterns of BmV-ATPase B in different sections of silk gland from the silkworm on the 3rd day of 5th instar and 1st day of wander stage by western blotting. BmV-ATPase B was expressed in all sections of the silk gland and it was abundant in the anterior silk gland (ASG) both in these two developmental stages. Furthermore, immunofluorescence indicated that BmV-ATPase B was located in the silk gland cells. Laser confocal scanning microscopy analysis revealed that BmV-ATPase B was mainly expressed in the cytomembrane of silk gland cells. These data elucidated the expression patterns of BmV-ATPase B in the silk gland of silkworm, which provides a good basis for further studies on the function of V-ATPase B in silk fiber formation.
Animals ; Bombyx ; enzymology ; Cloning, Molecular ; Escherichia coli ; metabolism ; Insect Proteins ; genetics ; metabolism ; Larva ; Recombinant Proteins ; genetics ; metabolism ; Silk ; Vacuolar Proton-Translocating ATPases ; genetics ; metabolism

OBJECTIVE: LASS2/TMSG1 gene is a novel tumor metastasis suppressor gene cloned from human prostate cancer cell line PC-3M in 1999 by Department of Pathology,Peking University of Basic Medical Sciences. It was found out that protein encoded by LASS2/TMSG1 could interact with the c subunit of vacuolar-ATPase (ATP6V0C). In this study, we explored the effect of LASS2/TMSG1 and its mutants on proliferation, migration and invasion of human prostate cancer cells and its molecular mechanism. METHODS: We constructed four LASS2/TMSG1 mutants and stably transfected the variants to human prostate cancer cell line PC-3M-1E8 cell with high metastatic potential. The stable transfectants were identified by qPCR and Western blot through analyzing the expression of LASS2/TMSG1 and ATP6V0C, the cell biology functions of LASS2/TMSG1 and its four mutants were studied using growth curve,MTT assay, soft agar colony formation assay, wound migration assay, Matrigel invasion study and flow cytometry. Furthermore, immunofluorescence was used to analysis the interaction of LASS2/ TMSG1 mutants and ATP6V0C. RESULTS: LASS2/TMSG1 mRNA and protein in LASS2/TMSG1 group and Mut1-Mut4 groups were higher than that in Vector group; Western blot showed that ATP6V0C protein in LASS2/TMSG1 wild group was lower than that in Vector group, but ATP6V0C protein in LASS2/TMSG1 S248A group was obviously higher than that in Vector group. MTT test and growth curve assay showed growth ability in LASS2/TMSG1 S248A group was increasing compared with other groups from day 5. Soft Agar colony formation experiment showed anchor independent growth ability in LASS2/TMSG1 S248A group was higher than those in the other groups (P<0.05), Cell migrations (from 35.3%±3.2% to 70.3%±3%) in LASS2/TMSG1 S248A group was increasing compared with LASS2/TMSG1 wild group (P<0.01), and more cells passed through Matrigel in LASS2/TMSG1 S248A group compared with LASS2/TMSG1 wild group (from 50±3.2 to 203±6.5, P<0.01), the apoptosis rate in LASS2/TMSG1 S248A group was obviously higher than that in LASS2/TMSG1 wild group (from 7% to 15.1%, P<0.05), and the G0/G1 ratio in LASS2/TMSG1 S248A group was obviously higher than that in LASS2/TMSG1 wild group (from 51.0% to 85.4%). Furthermore, double immunofluorescent staining observed the colocalization between ATP6V0C and LASS2/TMSG1 protein and its mutations, the expression of ATP6V0C in LASS2/TMSG1 S248A group increased significantly compared with the other groups. CONCLUSION LASS2/TMSG1 S248A promotes proliferation, migration and invasion of prostate cancer cells through increasing ATP6V0C expression, suggesting that aa248-250 is an important function site for LASS2/TMSG1 in invasion suppression of prostate cancer cells.
Beijing ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Humans ; Male ; Membrane Proteins/genetics* ; Mutation ; Neoplasm Invasiveness ; Prostatic Neoplasms/genetics* ; Sphingosine N-Acyltransferase/genetics* ; Transfection ; Tumor Suppressor Proteins/genetics* ; Vacuolar Proton-Translocating ATPases

OBJECTIVETo detect potential mutation of the TCIRG1 gene in a boy with infantile malignant osteopetrosis.

METHODSTarget sequence capture and next-generation sequencing were applied for the proband and his parents to identify the causative mutation, and Sanger sequencing was used to verify the suspected mutation.

RESULTSThe proband manifested at 4 months of age with symptoms including anemia, thrombocytopenia, hepatosplenomegaly, and cephalus quadratus. X-ray revealed generalized increased bone density. A novel compound heterozygous mutation, c.796G to T (p.E266X) and c.1372G to A (p.G458S), were identified in the boy. His father and grandmother also carried the c.796G to T (p.E266X) mutation, and his mother carried the c.1372G to A (p.G458S) mutation. Neither mutation was found in the PubMed and ClinVar databases.

CONCLUSIONThe novel compound heterozygous mutation c.796G to T (p.E266X) and c.1372G to A (p.G458S) probably underlies the disease in the proband. Above results may enrich the mutation spectrum of the TCIRG1 gene and provide new evidence for the molecular basis of infantile malignant osteopetrosis.

Adult ; Asian Continental Ancestry Group ; Base Sequence ; Humans ; Infant ; Infant, Newborn ; Infant, Newborn, Diseases ; genetics ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation ; Osteopetrosis ; genetics ; Pedigree ; Vacuolar Proton-Translocating ATPases ; genetics

OBJECTIVETo explore the application of preimplantation genetic diagnosis (PGD) for infantile malignant osteopetrosis (IMO).

METHODSFor a family affected with IMO, PGD was provided using combined parental mutation detection and haplotype constructions with microsatellite markers spanning the TCIRG1 gene. Prenatal diagnosis was performed on the chorionic villus and amniocentesis samples by direct sequencing.

RESULTSPrenatal diagnosis showed that the fetus by the third pregnancy has carried the parental mutations [c.242delC (p.Pro81Argfs*85) and c.1114C>T (p.Gln372*)], and the pregnancy was terminated. PGD was subsequently performed through mutations detection and haplotype analyses following whole genome amplification (WGA) of each of 13 cells. The results showed that 6 of the 13 embryos were unaffected, 3 were carriers and 4 were affected. Well developed unaffected/carrier embryos were selected and transferred into the uterus. A single pregnancy was confirmed. Subsequently pre- and post-natal diagnoses have confirmed development of a healthy child.

CONCLUSIONThe study demonstrated the advantage of PGD over prenatal diagnosis when natural pregnancies have repeatedly produced IMO children/fetuses.

Adult ; Base Sequence ; Female ; Fertilization in Vitro ; Fetus ; Genetic Carrier Screening ; Humans ; Infant ; Male ; Microsatellite Repeats ; Molecular Sequence Data ; Osteopetrosis ; diagnosis ; embryology ; genetics ; prevention & control ; Pedigree ; Point Mutation ; Pregnancy ; Preimplantation Diagnosis ; Vacuolar Proton-Translocating ATPases ; genetics

A novel vacuolar Na+/H+ exchanger, CgNHX1, was cloned from a halophytic species Chenopodium glaucum by using reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technique. Sequence alignment and phylogenetic analysis of 22 NHX genes from GenBank as well as the new CgNHX1 gene indicate that NHX genes shared a great degree of similarity, regardless of their glycophytic or halophytic origin. Expression of the CgNHX1 gene was induced by NaCl and peaked at 400 mmol/L NaCl. Overexpression of NHX1 genes in rice enhanced their tolerance to salt stress. However, there is no significant difference in salt tolerance among the transgenic rice plants overexpressing the NHX1 genes from either glycophytic or halophytic species. The Na+ content of both the wild type (WT) and transgenic plants increased when exposed to 50 and 100 mmol/L NaCl, and the Na+ concentration in transgenic plants was marginally higher than that of WT. Our data demonstrate that the overexpression of the NHX1 gene from either glycophytic or halophytic species resulted in the enhanced tolerance to salt stress at a similar level, suggesting that NHX gene per se might not be the reason accounting for the difference in salt tolerance between glycophytes and halophytes.
Amino Acid Sequence ; Cloning, Molecular ; DNA, Complementary ; metabolism ; Gene Expression Regulation, Plant ; Molecular Sequence Data ; Oryza ; genetics ; Phylogeny ; Plant Proteins ; genetics ; Plants ; genetics ; metabolism ; Plants, Genetically Modified ; Protein Structure, Tertiary ; Reverse Transcriptase Polymerase Chain Reaction ; Salts ; pharmacology ; Sequence Homology, Amino Acid ; Sodium Chloride ; pharmacology ; Vacuolar Proton-Translocating ATPases ; chemistry ; physiology

OBJECTIVETo explore effects of Yougui recipe (see text) and salmon calcitonin acetate in preventing osteolysis surrounding artificial prosthesis.

METHODSThirty-two SD male rats with weighted (250 +/- 20) g, aged 8 weeks, were randomly divided into four groups: blank group, model group, salmon calcitonin acetate group and Yougui recipe (see text) group, and 8 rats in each group. Blank group did not undergo any process, other 24 rats underwent anesthesia by chloral hydrate, their knee joints were exposed through medial patellar side,drilling from fermoral condyle nest to marrow cavity,high density of polythlene particles were injected into hole, titanium nail were put into, bone wax closed the window, then suturing step by step. After the molding, saline were used to gavaged in blank group and model group, Yougui recipe (see text) for Yougui recipe (see text) group, salmon calcitonin maximus injection for calcitonin group. After 10 weeks' mediation, rats were executed, and arterial blood and bilateral femoral organization were collected to biochemical, imaging morphology, tissue pathology and molecular biology detection.

RESULTSThe key gene expression of activiting osteoclast were inhibited in Yougui recipe (see text) group and calcitonin group. The level of OPG, Ca, ALP in Yougui recipe group were higher than calcitonin group (P<0.01); the content of RANKL were lower (P<0.01). There were no significance meaning in RANK, Trap5b, P between two groups.

CONCLUSIONBoth of Yougui recipe (see text) and calcitonin can slow and treat surrounding osteolysis of artificial joint prosthesis, and Yougui recipe (see text) has better effect in promoting bone formation. The effect of Yougui recipe (see text) in promoting bone formation, inhibiting osteoclasts to provide a new method to treating surrounding osteolysis of artificial joint prosthesis.

Animals ; Biomarkers ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation ; drug effects ; Interleukin-1 ; genetics ; Interleukin-6 ; genetics ; Male ; Osteolysis ; etiology ; metabolism ; pathology ; prevention & control ; Prostheses and Implants ; adverse effects ; Rats ; Rats, Sprague-Dawley ; Tomography, X-Ray Computed ; Vacuolar Proton-Translocating ATPases ; genetics

OBJECTIVETo explore the effects of LASS2/TMSG1 silencing on the growth, invasion and metastasis of prostate carcinoma cells and to investigate the related molecular mechanisms.

METHODSLASS2/TMSG1 expression of human prostate carcinoma cell line with low metastatic potentiality (PC-3M-2B4 cells) was knocked down using DNA vector-based small interfering RNA (shRNA), followed by evaluations of tumor cell invasion and metastasis.

RESULTSA stable PC-3M-2B4 cell line with expression of LASS2/TMSG1-shRNA was successfully established. MTT assay showed PC-3M-2B4 cells exhibited a strong proliferation after transfection of LASS2/TMSG1-shRNA.LASS2/TMSG1-shRNA transfected clones demonstrated an increased clonogenicity by soft agar colony formation assay and a significant increase of tumor cell invasion by matrigel invasion study.Flow cytometry showed that after LASS2/TMSG1 gene silencing, the apoptotic rate of PC-3M-2B4 cell significantly decreased (P<0.01) without significant cell cycle change (P>0.05).Eight weeks after implantation into subcutaneous tissues in BAL B/c (nu+) mice, the size and weight of sh-LASS2/TMSG1 xenografts were significantly larger than those of the control group (P<0.05).Nuclear proliferation index of the subcutaneous tumor was also higher in the LASS2/TMSG1 shRNA group than those in the control group. Lymph node metastasis was observed in 5 of 6 mice of LASS2/TMSG1 shRNA group and only 1 of 6 of the control group. V-ATPase activity, activities of secreted MMP-2 and MMP-9 and extracellular hydrogen ion concentration were significantly increased in LASS2/TMSG1-shRNA group compared with the control group (P<0.05).

CONCLUSIONSilencing of LASS2/TMSG1 promotes the growth, invasion and metastasis of prostate cancer cells through up-regulation of V-ATPase activity, indicating that LASS2/TMSG1 is a tumor metastasis suppressor gene.

Animals ; Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Gene Silencing ; Humans ; Hydrogen-Ion Concentration ; Lymphatic Metastasis ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; RNA, Small Interfering ; genetics ; Sphingosine N-Acyltransferase ; genetics ; metabolism ; Transfection ; Tumor Burden ; Tumor Suppressor Proteins ; genetics ; metabolism ; Up-Regulation ; Vacuolar Proton-Translocating ATPases ; metabolism