Analysis of genetic characteristics of 436 children with dysplasia and detailed analysis of rare karyotype.

Chromosomal abnormality is one of the important causes of dysplasia in children. However, due to regional and ethnic differences, the reported rates of chromosomal abnormalities in patients with dysplasia vary greatly. Moreover, the clinical manifestations in children with rare chromosomal diseases were heterogeneous. So, we retrospectively analyzed the karyotype results of 436 children with dysplasia and conducted a detailed analysis of rare chromosomal diseases. The results showed that chromosomal abnormalities were present in 181 of 436 cases. Intellectual disability, dysmorphology, congenital malformations, the disorder of sexual development, and short stature were the main five clinical symptoms in children with chromosomal abnormalities. Moreover, 136 cases of Trisomy 21 (Tri21) were detected, of which 130 were standard Tri21, 5 were robertsonian Tri21, and 1 was chimera type. In addition, 16 cases of rare abnormal karyotype, including complex Tri21, complex Turner syndrome, 4p-syndrome, 18q-syndrome, and 5p-syndrome, were also detected. In summary, chromosome abnormality is one of the important causes of dysplasia in children. Furthermore, prenatal screening and diagnosis could play a great significance in preventing dysplasia in children. In addition, the retrospective analysis of rare cases is valuable for clinical diagnosis and risk assessment of recurrence.

Cytogenetic analysis of 2959 couples with spontaneous abortion and detailed analysis of rare karyotypes.

Chromosome abnormality is one of the important causes of spontaneous abortion. However, due to regional and ethnic differences, the reported rates of chromosomal abnormalities in patients with spontaneous abortion vary greatly. At present, there is no large sample statistics of chromosome abnormality in patients with spontaneous abortion in Yantai, Shandong province, China and hence 2959 couples (5918 individuals) with spontaneous abortion were recruited for this study. G banding was used to examine the karyotype of patients. The results showed that chromosomal abnormalities were present in 173 of 2959 couples with the rate of 5.85%. Female carriers were significantly higher than male. Chromosomal abnormality rate was positively correlated with the number of spontaneous abortions. Structural aberrations were significantly greater than numerical aberrations, with a prevalence of 92.49% and 7.51%, respectively. Balanced translocation, Robertson translocation and inversion were the most common types of chromosomal structural abnormalities. Among them, the proportion of balanced translocation was the highest (63.13%, 101/160). In addition, three cases of rare complex abnormal karyotype were detected. In summary, chromosome abnormality could be one of the important causes of spontaneous abortion in Yantai, Shandong province, China. The sex of patients with chromosomal abnormalities and the number of spontaneous abortions should be considered in genetic counselling. When one of the partners have chromosome abnormality, preimplantation genetic diagnosis and prenatal diagnosis could play a great significance for preventing the birth of children with chromosomal diseases and reducing birth defects.

Clinical, cytogenetic and molecular analyses of a rare case with ring chromosome 15 and review of the literature.

OBJECTIVE: Ring chromosome 15 [r (15)], accompanied by a series of clinical symptoms, is a rare genetic disease. The genotype and phenotypic diversity of patients with r (15) still needed further enrichment. In this study we present a rare case of mosaic ring chromosome 15 with facial anomalies and extremities slenderness. CASE REPORT: This case involves a 30-year-old woman, unpregnancy within 6 years. Clinical examination of the patient only revealed facial anomalies and extremities slenderness. The result of routine G-band karyotyping was 46,XX,r(15)(p12q26.3)[53]/46,XX,r(15;15)(p11.2q26.3;p11.2q11.2)[28]/45,XX, -15[10]/46,XX,r(15;15)(p11q26.3;p11q26.3)[4]. SNP array was employed to investigate the genome copy number variations (CNVs). The result revealed that there was a micro-duplication of 2.0 Mb at 15q26.3(arr[ph19]15q26.3 (100,400,214- 102,429,112)×3). The duplicated chromosomal section encompassed genes including CHSY1, ALDHIA3, LRRK1, and INS1. We further compared to the cytogenetic characteristics and clinical symptoms of the patient with those already reported by reviewing the literature. CONCLUSION: This report is especially helpful to supplement the phenotypic diversity of patients with r (15).

Molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from chromosome 8 associated with congenital hypoplasia of the tongue and review of the literature.

OBJECTIVE: To present molecular cytogenetic characterization of mosaic supernumerary ring chromosome 8 which has trisomy of a region of chromosome 8p12-q21.13 associated with congenital hypoplasia of the tongue and review of the literature. CASE REPORT: A 27 year-old woman presented with congenital hypoplasia of the tongue. The chromosome karyotype of peripheral blood lymphocytes was detected by conventional cytogenetic analysis. The genome copy number variations were detected by SNP array. Conventional cytogenetic analysis of the peripheral blood revealed a karyotype of 47,XX,+mar[60]/46,XX[40]. SNP array revealed that there was a duplication of 45.2 Mb at arr[hg19] 8p12q21.13(36,013,636-81,263,140) × 2-3. CONCLUSION: With this study a patient involving mosaic trisomy 8p12-q21.13 along with clinical properties, is described and compared to previously reported cases involving a small supernumerary marker chromosome (sSMC) derived from chromosome 8.

Biomechanical effects of pedicle screw adjustments on the thoracolumbar burst fractures / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Posterior pedicle screw device is widely used in treatment of thoracolumbar burst fractures. As the clinical operation is not based upon quantitative data of adjustments, the results are not optimal. At present, no study has assessed the associations between the device adjustments and the restoration of stiffness. We investigated the biomechanical effects that adjustments of a pedicle screw device had on the burst fracture, and explored an optimal adjustment.</p><p><b>METHODS</b>Burst fractures were produced at L1 vertebra in 24 fresh calf spines (T12-L3). The specimens were divided into four groups at random. Pedicle screw devices were attached to T13 and L2. Four device adjustments, consisting of distraction and extension, were applied. Adjustment 1 was pure 6° extension, adjustment 2 was pure 5 mm distraction, adjustment 3 was 6° extension followed by 5 mm distraction, and adjustment 4 was 5 mm distraction followed by 6° extension. The effect of each adjustment on the stiffness restoration, anatomical reduction, and neural decompression for the burst fractures was analyzed and evaluated.</p><p><b>RESULTS</b>Pure extension (Group 1) produced the closest segment height and the least restoration of the canal to the intact. Pure distraction (Group 2) restored stiffness most, but with only 60% stiffness of the intact value, and lost the segmental angle most to the intact. The combination of extension-distraction (Group 3 and Group 4) produced the maximum reduction of the anatomy and restoration of the canal in the burst fracture, and the least stiffness restoration. The sequence of extension and distraction did not affect stiffness restoration, anatomical reduction, and neural decompression.</p><p><b>CONCLUSIONS</b>The device adjustments affected stiffness restoration, anatomical reduction, and neural decompression. The combined extension-distraction adjustment may be the most suitable considering the anatomical reduction and neural decompression, but the stiffness decreased the most; it should be considered to reconstruct L1 vertebra.</p>

Monoclonal antibodies against nucleophosmin mutants: potentials for the detection of acute myeloid leukemia.

Nucleophosmin (NPM1) gene mutations resulting in cytoplasmic delocalization of Nucleophosmin (NPMc+) are the most common genetic alteration in acute myeloid leukemia (AML). Here, we attempted to prepare monoclonal antibodies (mAbs) against NPM1 mutation A (NPM-mA) and investigated the mAbs' clinical utility in immunohistochemical detection of NPMc+AML. The pET-32a-NPM-mA vector with the whole open reading frame of the NPM-mA gene was constructed. E.coli BL21 transformed with the vector were induced to express the NPM-mA recombinant protein. BALB/c mice were immunized with the recombinant NPM-mA. Positive clones were selected by indirect ELISA and the mAbs were obtained. Immunohistochemistry was performed to detect the NPMc+ in bone marrow smears from 10 AML patients with NPM-mA. The results showed that the pET-32a-NPM-mA vector was successfully constructed and the NPM-mA recombinant protein was used to immunize the mice. Two positive clones (2G3 and 3F9) were selected. The mAbs against NPM-mA were raised, but did cross-react with wild type NPM1. The mAbs can be used to detect the cytoplasmic dislocation of NPM1 in all AMLs carrying NPM-mA. Our results show that anti-NPM-mA mAbs were produced. Though they would cross-react with wild type NPM1, the mAbs may still have potential in the detection of NPMc+AMLs.

Knockdown of NPM1 by RNA interference inhibits cells proliferation and induces apoptosis in leukemic cell line.

Nucleophosmin (NPM1) is an abundant and ubiquitously expressed phosphoprotein that is known to influence solid tumors progression. However, little is known about the role of NPM1 in leukemia. Here, we knocked down the NPM1 expression by RNA interference to investigate the role of NPM1 in leukemic cells proliferation and apoptosis. The interference vector pNPM1-shRNA was constructed and transfected into the human leukemic K562 cell line. The expression levels of NPM1 mRNA and protein were detected by quantitative real-time PCR and Western blot, respectively. Cells proliferation potential in vitro was assessed by methyl thiazolyl tetrazolium (MTT) and colony formation assays. Flow cytometry was used to detect the distribution of cell cycle. Cellular apoptosis was reflected by the relative activities of caspase-3 and caspase-8. The results showed that the expression levels of NPM1 mRNA and protein in K562 cells were significantly reduced after pNPM1-shRNA transfection. The cells growth was significantly inhibited in a time-dependent manner and the number of colonies was significantly reduced in the pNPM1-shRNA transfected cells. Meanwhile, the percentage of cells in G1 phase in the K562/pNPM1-shRNA cells was significantly increased. In addition, there were higher relative activities of caspase-3/8 in the pNPM1-shRNA transfected cells. These results indicate that down-regulation of NPM1 expression inhibits leukemic cells proliferation, blocks cell cycle progression and induces cellular apoptosis. It may implicate a potential target for leukemia gene therapy.

Nucleophosmin gene mutations promote NIH3T3 cell migration and invasion through CXCR4 and MMPs.

Nucleophosmin (NPM1) plays key roles in ribosome biogenesis, centrosome duplication, and maintenance of genomic integrity. NPM1 mutations have been recently identified as the most frequent genetic alteration in acute myeloid leukemia and are related to leukemogenesis. NPM1 mutations are involved in the regulation of cell proliferation, cell cycle, and apoptosis. However, the oncogenic potential of NPM1 mutations is not fully understood. Here, we investigated the change of cell migration and invasion in vitro and the role of NPM1 mutations in this process. In our study, NIH3T3 cells were transfected with plasmids encoding NPM1 mutation A (NPM1 mA), and the cell chemotactic response in vitro was evaluated by cell migration and invasion assays. In addition, the expression levels of MMP-2, MMP-9 and CXCR4 were assayed by quantitative real-time PCR and western blotting. Our findings suggested that the migration and invasion of NIH3T3 cells were significantly enhanced after transfection with NPM1 mA (p<0.01). Furthermore, there was greater expression of MMP-9 and CXCR4 (p<0.01), but a lower expression of MMP-2 in the NPM1 mA group. These results demonstrate that NPM1 mutations may promote cell migration and invasion in vitro, and MMP-9 and CXCR4 may be involved in the regulation of cell invasion. Thus, this study sheds new light on the effect of NPM1 mutations on leukemogenesis.

Increased integrity of circulating cell-free DNA in plasma of patients with acute leukemia.

BACKGROUND: Increased cell-free DNA (cf-DNA) and the integrity of cf-DNA in plasma of patients with cancer has been described. We investigated the clinical utility of cf-DNA in the detection and monitoring of progression of leukemia. METHODS: Plasma samples from 60 patients with acute leukemia were analyzed in comparison to plasma from 30 healthy controls. Plasma DNA was determined by quantitative PCR (qPCR) by amplifying the ß-actin gene (ACTB). The DNA integrity index was calculated as the ratio of qPCR results (ACTB384/106). Paired diagnostic/complete remission (CR)/relapse samples from eight of 60 patients were analyzed, and the minimum residual disease (MRD) situations were monitored. RESULTS: DNA concentrations (median: 8.80 ng/mL, p=0.004) and DNA integrity (median: 0.51, p<0.001) in cancer patients were significantly higher. Receiver operating characteristic (ROC) curve analysis showed that the area under the ROC curve of DNA and DNA integrity were 0.79 and 0.88, respectively. DNA integrity at CR had a distinct reduction and then an increase at relapse. DNA integrity in CR cases was higher than that observed in healthy controls. CONCLUSIONS: Our preliminary data suggest that plasma DNA integrity is increased in acute leukemia and may be a potential biomarker for monitoring MRD. However, more work is needed.

Fluorouracil selectively enriches stem-like leukemic cells in a leukemic cell line.

Recent studies have reported that cancer stem cells (CSCs) could be isolated from solid cancer cell lines, in which the purity of CSCs was higher than that from tumor tissues. Separation of CSCs from leukemic cell lines was rarely reported. In this study, CD34(+)CD38(-)stem-like cell subsets in human KG-1a leukemic cell line were enriched by cytotoxic agent 5-fluorouracil (5-FU). After 4 days incubation of KG-1a cell line with 5-FU (50 microg/ml), the CD34(+)CD38(-) subpopulation of cell lines was enriched more than 10 times. The enriched cells had proliferate potential in vitro, low level of RNA transcription and Hoechst 33342 dye efflux ability, accompanied by high expression of ATP-binding cassette transporter protein ABCG2. Our findings suggest that treatment with 5-FU offers an easy method to isolate leukemic stem-like subpopulation. It can facilitate studies of leukemic stem cell biology and the development of new therapeutic strategies.