Assessment of intestinal status in MPLW515L mutant myeloproliferative neoplasms mice model.

The MPLW515L mutation is a prevalent genetic mutation in patients with myeloproliferative neoplasms (MPN), and utilizing this mutation in mice model can provide important insights into the disease. However, the relationship between intestinal homeostasis and MPN mice model remains elusive. In this study, we utilized a retroviral vector to transfect hematopoietic stem cells with the MPLW515L mutation, creating mutated MPN mice model to investigate their intestinal status. Our results revealed that the MPLW515L in MPN mice model aggravated inflammation in the intestines, decreased the levels of tight junction proteins and receptors for bacteria metabolites. Additionally, there was increased activation of the caspase1/IL-1ß signaling pathway and a significant reduction in phos-p38 levels in the intestinal tissue in MPN mice. The MPLW515L mutation also led to up-expression of anti-microbial genes in the intestinal tract. Though the mutation had no impact on the alpha diversity and dominant bacterial taxa, it did influence the rare bacterial taxa/sub-communities and consequently impacted intestinal homeostasis. Our findings demonstrate the significance of MPLW515L mice model for studying MPN disease and highlight the mutation's influence on intestinal homeostasis, including inflammation, activation of the IL-1ß signaling pathway, and the composition of gut microbial communities.

Inhibition of PAK1 generates an ameliorative effect on MPLW515L mouse model of myeloproliferative neoplasms by regulating the differentiation and survival of megakaryocytes.

Most thrombopoietin receptor (MPL) mutations result in abnormal megakaryocyte expansion in the spleen or bone marrow (BM), leading to progressive fibrosis. It has been reported that p21 (Rac Family Small GTPase 1 [RAC1])-activated kinase 1 (PAK1) participates in the proliferation and differentiation of megakaryoblasts. PAK1 phosphorylation increased in patients with myeloproliferative neoplasms (MPNs) and murine MPN cells with the Mplw515l mutant gene in this study; however, the function of overactivated PAK1 in MPN cells remains unclear. We found that inhibition of PAK1 caused significant changes in the biological behaviors of MPLW515L mutant cells in vitro, including arrested growth or reduced clonality and increased polyploid DNA and cell apoptosis due to upregulated cleaved caspase 3. In vivo, PAK1 inhibitor treatment caused a slow elevation of leukocytosis and hematocrit (HCT) and a reduction in hepatosplenomegaly in 6133/MPLW515L-transplanted mice, along with reduced tumor cell infiltration and prolonged survival. Further, deletion of PAK1 sustained a relatively normal HCT and platelet count at the beginning of the disease but did not completely alleviate the splenomegaly of MPLW515L mutant mice. Notably, PAK1 knockout attenuated the destruction of splenic structure, and reduced the megakaryocyte burden within the BM. These results suggest that inhibition of PAK1 may be a useful method for treating MPLW515L mutant MPN by intervening megakaryocytes.

[Construction of a Mouse Model for Myeloproliferative Neoplasms and an Evaluation System].

OBJECTIVE: To construct a myeloproliferative neoplasms (MPN) transplanted mouse model with JAK2-V617F, MPLW515L or CALR-Type I gene mutation, and establish a systematic evaluation system to verify the success of model construction. METHODS: The bone marrow c-kit+ cells of the mice were obtained by the following steps: The mice were killed by cervical dislocation, the femur, tibia and ilium were separated, and the bone marrow cells were collected. The c-kit+ cells were sorted after incubation with CD117 magnetic beads. The method of constructing mouse primary mutant cells is as follows: A gene mutation vector with a GFP tag was constructed by the retroviral system, and the retroviral vector was packaged into the Platinum-E cells to obtain the virus supernatant, and then used it to infect the c-kit+ cells of mice. The MPN mouse model was constructed as follows: the mouse primary c-kit+ cells containing the mutant genes were collected after infection, and then transplanted them via the tail vein into the female recipient mice of the same species which were irradiated with a lethal dose of gamma rays (8.0 Gy). The MPN mouse model was evaluated as follows: After transplantation, the peripheral blood of the mice was regularly collected from the tail vein to perform the complete blood count test, and the size of spleen and the degree of bone marrow fibrosis were estimated. RESULTS: The mouse c-kit+ cells with the mutant genes were successfully obtained from the bone marrow. MPN mouse model was successfully constructed: The peripheral blood cells of the MPN-transplanted mice carried exogenous implanted GFP-positive cells, and the white blood cells (WBC), platelet (PLT) and hematocrit (HCT) were all increased; the body weight loss, and the water and food intake were reduced in the transplanted mice; further pathological analysis showed that the transplanted mice displayed splenomegaly and bone marrow fibrosis. These results suggested that the MPN mouse model was successfully constructed. According to the common and different characteristics of the three MPN mouse model, a preliminary evaluation system for judging the success of MPN mouse model construction was summarized, which mainly included the following indicators, for example, the proportion of GFP-positive cells in the peripheral blood of mice; WBC, PLT and HCT; the degree of spleen enlargement and the bone marrow fibrosis. CONCLUSION: The MPN mouse model with JAK2-V617F, MPLW515L or CALR-Type I gene mutation is successfully established by retroviral system, which can provide an important experimental animal model for the research of MPN pathogenesis and drug-targeted therapy.

[Identification of two novel variants of the PCCB gene in a pedigree affected with propionic acidemia].

OBJECTIVE: To detect pathogenic variants in a pedigree affected with propionic acidemia (PA). METHODS: The proband was subjected to high-throughput next-generation sequencing. Suspected variants were validated by Sanger sequencing of his family members. mRNA was extracted from peripheral blood lymphocytes from the proband's father in order to verify the impact of the splicing variant by RT-PCR combined with Sanger sequencing. The pathogenicity of the missense variant was predicted by using PolyPhen-2, Mutation Taster, SIFT, COBALT and HOPE software. RESULTS: The proband was found to harbor compound heterozygous variants of the PCCB gene, namely c.184-2A>G and c.733G>A (p.G245S), which were respectively inherited from his father and mother. RT-PCR combined with Sanger sequencing confirmed skipping of exon 2 during transcription. Bioinformatic analysis indicated the c.733G>A (p.G245S) variant to be damaging. CONCLUSION: The two variants of the PCCB gene probably underlay the disease in this patient. Above findings have enriched the spectrum of PCCB gene variants.

[Analysis of ACAT1 gene variants in a patient with ß-ketothiolase deficiency].

OBJECTIVE: To explore the genetic etiology of a child suspected for ß-ketothiolase deficiency by neonatal screening. METHODS: All coding exons and flanking sequences of the ACAT1 gene were subjected to targeted capture and high-throughput sequencing. Suspected variants were verified by Sanger sequencing and bioinformatic analysis. RESULTS: The child was found to harbor compound heterozygous variants of the ACAT1 gene, namely c.121-3C>G and c.275G>A (p. Gly92Asp). The c.121-3C>G variant was also detected in his father and two sisters, while the c.275G>A (p. Gly92Asp) was a de novo variant. A c.334+ 172C>G (rs12226047) polymorphism was also detected in his mother and two sisters. Sanger sequencing has verified that the c.275G>A (p. Gly92Asp) and c.334+172C>G (rs12226047) variants are located on the same chromosome. Bioinformatics analysis suggested both c.121-3C>G and c.275G>A (p.G92D) variants to be damaging. Based on the American College of Medical Genetics and Genomics standards and guidelines, the c.275G>A variant of the ACAT1 gene was predicted to be pathogenic (PS2+ PM2+ PM3+ PP3+PP4), the c.121-3C>G variant to be likely pathogenic (PM2+ PM3+ PP3+PP4). CONCLUSION: The c.121-3C>G and c.275G>A variants of the ACAT1 gene probably underlay the pathogenesis of the child. Above finding has enriched the variant spectrum of the ACAT1 gene.

Long Noncoding RNA MALAT1 Knockdown Inhibits Proliferation, Migration, and Invasion and Promotes Apoptosis in Non-Small-Cell Lung Cancer Cells Through Regulating miR-515-3p/TRIM65 Axis.

Background: Long noncoding RNAs (lncRNAs) and mRNAs (messenger RNAs) have been reported to exert function in non-small-cell lung cancer (NSCLC), but how lncRNAs and mRNAs operate in the regulation of NSCLC is unclear. The purpose of this research was to elucidate the functional mechanism of lncRNA metastasis associated in lung adenocarcinoma transcript 1 (MALAT1) and tripartite-motif protein family member 65 (TRIM65) in NSCLC. Materials and Methods: Quantitative real-time polymerase chain reaction and western blot assay were employed to measure gene expression. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry analysis were performed to assess cell proliferation and apoptosis, respectively. Also, cell migratory and invasive abilities were detected by transwell assay. The interaction between miR-515-5p and MALAT1 or TRIM65 was predicted by starBase and then confirmed with the dual luciferase reporter assay, RNA immunoprecipitation (RIP) assay, or pull-down assay. Besides, mouse xenograft was conducted to analyze the effect of MALAT1 knockdown on tumor growth in vivo. Results: MALAT1 and TRIM65 expression was upregulated, and miR-515-5p expression was downregulated in NSCLC tissues and cells. Both MALAT1 knockdown and TRIM65 depletion suppressed cell proliferation, migration, and invasion and induced apoptosis in NSCLC cells. Interestingly, MALAT1 directly inhibited miR-515-5p expression and miR-515-5p decreased TRIM65 level through interaction. MALAT1 knockdown repressed NSCLC cell growth via modulation of miR-515-5p/TRIM65 axis. Furthermore, silencing MALAT1 inhibited tumor growth in vivo. Conclusions: Our findings demonstrated that MALAT1 depletion inhibited the growth of NSCLC cells by regulating miR-515-5p/TRIM65 axis, providing the theoretical basis for the therapy of NSCLC.

RFC3 induces epithelial­mesenchymal transition in lung adenocarcinoma cells through the Wnt/ß­catenin pathway and possesses prognostic value in lung adenocarcinoma.

Lung cancer is a malignant tumor responsible for the highest mortality rate in humans. The identification of novel functional genes is of great importance in the treatment of lung cancer. The reported roles of replication factor C subunit 3 (RFC3) in tumorigenesis are contradictory. The present study aimed to explore the role and mechanism of RFC3 in lung cancer cells. An immunohistochemical study of 165 lung cancer and adjacent tissues was conducted (123 lung adenocarcinoma tissues and 42 lung squamous cell carcinoma tissues). Kaplan­Meier analysis and Cox multivariate analysis were employed to explore the relationship between RFC3 and patient prognosis. In addition, the proliferation, cell cycle distribution and apoptosis of A549 and H1299 cells were determined by MTT assay and flow cytometry, respectively, following cell transfection to induce overexpression and knockdown of RFC3. A Boyden chamber assay and wound­healing assay were conducted to determine the invasive and migratory abilities of A549 and H1299 cells. Western blotting was used to analyze the effects of RFC3 overexpression and RFC3 small interfering RNA­induced knockdown, and to explore the potential mechanism and pathway underlying the effects of RFC3. Positive expression of RFC3 was detected in lung adenocarcinoma, and overexpression of RFC3 shortened the survival time of patients with lung adenocarcinoma. Furthermore, overexpression of RFC3 increased the invasion and migration of A549 cells, whereas knockdown of RFC3 significantly reduced the invasion and migration of H1299 cells. Ectopic expression of RFC3 induced epithelial­mesenchymal transition (EMT), as determined by downregulation of E­cadherin, and upregulation of N­cadherin, vimentin and Wnt signaling target genes, including c­MYC, Wnt1 and ß­catenin, and the ratio of phosphorylated­glycogen synthase kinase 3 (GSK3)­ß (Ser9)/GSK3­ß. In conclusion, RFC3 may be considered a coactivator that promotes the Wnt/ß­catenin signaling pathway, and induces EMT and metastasis in lung adenocarcinoma.

Long non-coding RNA DLEU1 exerts an oncogenic function in non-small cell lung cancer.

OBJECTIVE: To explore the role of long non-coding RNA DLEU1 in the tumorigenesis and progression of non-small cell lung cancer (NSCLC) and the underlying mechanisms. METHODS: The expression of DLEU1 in NSCLC tumor tissues and adjacent normal tissues was evaluated using bioinformatics analysis and qPCR. The effects of DLEU1 overexpression or deficiency on cell proliferation, apoptosis, migration and invasion were explored experimentally. Additionally, the impact of DLEU1 up-regulation on tumourigenesis was also assessed in vivo. RESULTS: The expression of DLEU1 was up-regulated in NSCLC tumor tissues. DLEU1 overexpression promoted the proliferation, migration, and invasion, but inhibited the apoptosis of NSCLC cells by upregulating CDK1 expression, binding with SRC and altering the expression of P70(S6K), MMP2 and E-cadherin. Besides, xenograft tumors in nude mice demonstrated that DLEU1 overexpression accelerated tumor growth. CONCLUSIONS: DLEU1 promoted tumorigenesis and progression of NSCLC, and might be a promising therapeutic target for NSCLC.

[Analysis of PHEX gene mutations in three pedigrees affected with hypophosphatemic rickets].

OBJECTIVE: To explore the molecular basis for three pedigrees affected with hypophosphatemia vitamin D resistant rickets (X-linked hypophosphatemia, XLH). METHODS: Peripheral blood samples from the three pedigrees were collected. Following DNA extraction, the 11 exons and flanking regions of the PHEX gene were subjected to PCR amplification and direct sequencing. Pathogenicity of identified mutations was evaluated through genotype-phenotype correlation. RESULTS: For pedigrees 1 and 2, pathogenic mutations were respectively identified in exon 8 (c.871C>T, p.R291X) and exon 15 (c.1601C>T, p.P534L) of the PHEX gene. For pedigree 3, a novel mutation (c.1234delA, p.S412Vfs*12) was found in exon 11 of the PHEX gene, which caused shift the reading frame and premature termination of protein translation. CONCLUSION: The three mutations probably account for the XLH in the affected pedigrees. The discovery of novel mutations has enriched the spectrum of PHEX gene mutations.

[Analysis of clinical manifestation and genetic mutations in two patients with Cornelia de Lange syndrome].

OBJECTIVE: To detect potential mutations in two neonates suspected for Cornelia de Lange syndrome (CdLS). METHODS: Peripheral blood samples from the neonates and their parents were collected and analyzed for CdLS-related genes using targeted sequence capture and next-generation sequencing. Suspected mutations were confirmed by direct Sanger sequencing. RESULTS: The neonates were found to respectively carry mutations c.7219C to T and p.D2339Lfs*4 of the NIPBL gene, among which the p.D2339Lfs*4 mutation has not been reported previously. No pathogenic mutation was found in other CdLS-related genes including NIPBL, SMC1A, SMC3, RAD21 and HDAC8. CONCLUSION: The c.7219C to T and p.D2339Lfs*4 mutations of the NIPBL gene probably account for the disease in both patients.

Inhibition of ITCH Suppresses Proliferation and Induces Apoptosis of Lung Cancer Cells.

BACKGROUND/AIMS: The E3 ubiquitin ligase ITCH plays an important role in invasive and metastatic cancers. However, the role of ITCH in the progression of lung cancer has not been fully described. METHODS: Real-time PCR was used to detect the expression of ITCH mRNA in the tumor tissues and paracarcinoma tissues from 32 patients with lung cancer. SiRNA was used to inhibit the expression of ITCH in two lung cancer cell lines, H1975 and Calu3 and the cell proliferation and apoptosis were measured by MTT and flow cytometric assay. In addition, to further investigate whether ITCH affected the apoptosis of cancer cells and its underlying mechanisms, the expression of important markers of apoptosis and invasion in lung cancer cells were detected by Western blot. RESULTS: The study showed significant increments in the expression of ITCH in lung cancer tissues (p< 0.001). ITCH siRNA effectively inhibited the proliferation and invasion of the lung cancer cells and promoted cell apoptosis. Molecular analysis further showed significant reductions in the expression of Bcl2, MMP2, MMP9 and ß-catenin and an increase in the expression of Bax and E-cadherin in the lung cancer cells with ITCH deficiency. CONCLUSIONS: Inhibition of ITCH might suppress lung cancer proliferation and invasion via regulation of MMPs, EMT and Bcl2/Bax signaling pathway.

[Detection of a patient with ring chromosome 15 by low-coverage massively parallel copy number variation sequencing].

OBJECTIVE: To explore the genetic cause for a child with developmental delay. METHODS: The karotypes of the child and her parents were analyzed with G-banding analysis. Their genome DNA was analyzed with low-coverage massively parallel copy number variation sequencing (CNV-seq) and verified by single nucleotide polymorphism array (SNP-array). RESULTS: The karyotype of the child was ascertained as 46,XX,r(15)(p13q26.3), while both parents showed a normal karyotype. CNV-seq and SNP-array have identified a de novo 15q26.2-q26.3 deletion in the child with a size of approximately 3.60 Mb. CONCLUSION: The abnormal phenotype of the patient carrying the ring chromosome 15 may be attributed to the presence of the 15q26.2-q26.3 microdeletion. The deletion and haploinsufficiency of the IGF1R gene probably underlie the main clinical features of the patient.

Invasion of trophoblast cell lines is inhibited by miR-93 via MMP-2.

Preeclampsia (PE) is a serious pregnancy-related syndrome, which is characterized by gestational hypertension and proteinuria. The microRNA-93 (miR-93) is upregulated in the maternal plasma of patients with PE. However, the functional role of miR-93 in PE remains unknown. Here, we identified that miR-93 inhibits trophoblastic invasion, which is correlated with PE development. In immortalized trophoblast cell lines, transwell assay showed that miR-93 mimics significantly inhibited the migration and invasion of immortalized trophoblast cells, whereas miR-93 inhibitors significantly promoted cell migration and invasion. Moreover, luciferase assays confirmed that miR-93 directly bound to the 3'untranslated region of matrix metalloproteinase-2 (MMP-2), and western blotting showed that miR-93 suppressed the expression of MMP-2 at the protein levels. This study indicated that miR-93 inhibits MMP-2 and reduces migration and invasion of immortalized trophoblast cells. Thus, miR-93 may represent a potential therapeutic target for PE intervention.

Clinical significance of Wip1 overexpression and its association with the p38MAPK/p53/p16 pathway in NSCLC.

Wip1 is deregulated in numerous human malignancies. However, its roles in non­small cell lung cancer (NSCLC) remain unclear. In the current study, the expression of Wip1 was investigated in NSCLC and its clinical significance was detected. Immunohistochemical staining was used to measure the expression of (wild­type p53 induced phosphatase 1) Wip1, p38 mitogen­activated protein kinase (MAPK), p53, p16 protein in a group of 60 NSCLC and 20 normal lung tissues. In addition, western blotting was performed to detect the Wip1 protein in fresh tissues. The correlations between clinical characteristics and Wip1 expression were analyzed using SPSS, version 16.0 software. The expression of Wip1 was positive in 63.3% (38/60) of NSCLC tissues, and in none of the normal lung tissues (0/20; P<0.01). In addition, Wip1 overexpression was significantly associated with tumor length and differentiation (P=0.008 and 0.03, respectively). The expression of Wip1 was negatively correlated with that of p38MAPK, p53 and p16 (r=­0.284, ­0.352 and ­0.348, respectively). The results of the current study demonstrated that Wip1 was frequently overexpressed in NSCLC, which may serve an essential role in the p38MAPK/p53/p16 signaling pathway.

Thymic carcinoma patients with myasthenia gravis exhibit better prognoses.

BACKGROUND: Thymic carcinoma is an aggressive mediastinal neoplasm with a poor prognosis, but the factors that contribute to its prognosis are not completely understood. Myasthenia gravis (MG) can coexist with thymic carcinoma with low incidence, but the effect MG has on thymic carcinoma prognosis is unknown. Here, we investigated the prognostic factors of thymic carcinoma and the influence of MG on patients with this disease. METHODS: Between January 1996 and December 2012, 49 patients were diagnosed with thymic carcinoma and surgically treated at our institution. Clinical data and survival information were recorded and systematically reviewed. Kaplan-Meier survival curves were generated, and independent prognostic factors were identified by Cox's proportional hazard model. RESULTS: Complete resection was achieved in 30 patients (61.2 %), and incomplete resection was performed on the other 19 patients (38.8 %). Six of the 49 patients with thymic carcinoma also presented with MG (12.2 %). Interestingly, these 6 patients exhibited much better prognoses when compared to the other 43 patients. Patients with MG also had significantly smaller tumors (P = 0.045), earlier Masaoka stage (P = 0.048), and higher complete resection rates (P = 0.042). However, multivariate analysis demonstrated that complete resection was the only independent predictor for overall survival (OS) (P < 0.001). CONCLUSIONS: The OS of patients with thymic carcinoma depends on complete resection, but patients with MG also demonstrate improved prognoses. MG patients have higher rates of complete surgical resection, which may account for their better prognoses. Patients with MG have unique features that may aid in the clinical management of these patients.

Role of AXL expression in non-small cell lung cancer.

The present study aimed to investigate the expression profile of AXL in non-small cell lung cancer (NSCLC) and its clinical significance. The current study included 257 NSCLC patients, tyrosine-protein kinase receptor UFO (AXL) expression in paired lung cancer and adjacent normal lung tissues of NSCLC patients were compared by immunohistochemistry, western blot analysis and quantitative polymerase chain reaction (qPCR). These methods were used to detect the expression of the AXL gene and protein in fresh tissues from 35 patients. Small interfering RNA (siRNA) was transfected into the H1299 lung cancer cell line to knock down AXL expression; the effects of AXL-siRNA on cell proliferation and migration were examined by MTT and Transwell migration assay, respectively. It was found that AXL staining density in lung cancer tissues was significantly increased compared with adjacent normal lung tissues (55.25 vs. 26.85%; P<0.01); and the expression level of AXL in NSCLC patients was significantly associated with the degree of tumor differentiation (P<0.01) and the clinical stage of disease (P<0.01). Western blotting and qPCR showed that AXL expression was significantly higher in cancer tissues compared with that in adjacent lung tissue (P<0.05). Additionally, the current study also showed that AXL-siRNA inhibited H1299 cell proliferation and migration in vitro. The present study demonstrates the association between increased expression of AXL in NSCLC and the low differentiation phenotype, and its effects on cell proliferation and migration, suggesting its potential clinical values for the prognosis of NSCLC.

Insights into the roles of hnRNP A2/B1 and AXL in non-small cell lung cancer.

Lung cancer has long been one of the most serious types of malignant tumor, and is associated with high incidence and mortality rates. Despite advancements in the comprehensive treatment of the disease, particularly with targeted therapeutic agents, there has been little improvement in the 5-year survival rates of patients. One of the leading causes of mortality in lung cancer is the lack of effective early diagnostic criteria. On this basis, the present study aimed to identify an index with potential in the early diagnosis and prognosis of lung cancer. The current study determined the expression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 and AXL proteins in non-small cell lung cancer (NSCLC) tumor samples, and performed prognostic analysis of the collected clinical data to identify any association. In addition, RNA interference was performed to silence the expression of hnRNP A2/B1, allowing evaluation of its molecular and cellular functions, and determination of the mechanism of hnRNP A2/B1 in NSCLC by means of AXL mediation. It was identified that the positive expression rate of hnRNP A2/B1 and AXL proteins were significantly higher in NSCLC compared with paracancerous lung tissues (P<0.05). Furthermore, the expression of hnRNP A2/B1 protein was correlated with the expression AXL. Thus, the expression of hnRNP A2/B1 and AXL protein are factors affecting prognosis in patients with NSCLC. Of these, hnRNP A2/B1 appears to be an independent risk factor.

PIWIL2 promotes progression of non-small cell lung cancer by inducing CDK2 and Cyclin A expression.

BACKGROUND: PIWI proteins have important roles in tumorigenesis due to their interaction with piRNAs. Recent studies suggest that PIWI proteins affect prognosis of various cancers. METHODS: In the present study, PIWI genes expression was assayed in non-small cell lung cancer (NSCLC). To determine the effects of PIWIL2 on NSCLC cells, overexpression and interference assays were performed using the A549 and H460 cell lines. The tumor formation model was performed to demonstrate the effects of PIWIL2 on tumor formation in vivo. RESULTS: PIWIL2 was increased both at the RNA and protein level in malignant cancer tissues compared with adjacent normal tissue. Moreover, increased PIWIL2 gene expression was negatively correlated with prognosis in NSCLC patients. Overexpression and interference of PIWIL2 promoted and depressed cell proliferation, respectively. Meanwhile, PIWIL2 interference arrested cells at the G2/M stage. In addition, we found that CDK2 and Cyclin A expression were correlated with PIWIL2 expression. Moreover, transfection of PIWIL2 promoted tumor growth in nude mice. CONCLUSION: Our findings shed light on the function of PIWIL2 in NSCLC and suggest potential prognostic and therapeutic value.

[Molecular and prenatal diagnosis of a family with Fanconi anemia by next generation sequencing].

OBJECTIVE: To provide prenatal diagnosis for a pregnant woman who had given birth to a child with Fanconi anemia with combined next-generation sequencing (NGS) and Sanger sequencing. METHODS: For the affected child, potential mutations of the FANCA gene were analyzed with NGS. Suspected mutation was verified with Sanger sequencing. For prenatal diagnosis, genomic DNA was extracted from cultured fetal amniotic fluid cells and subjected to analysis of the same mutations. RESULTS: A low-frequency frameshifting mutation c.989_995del7 (p.H330LfsX2, inherited from his father) and a truncating mutation c.3971C>T (p.P1324L, inherited from his mother) have been identified in the affected child and considered to be pathogenic. The two mutations were subsequently verified by Sanger sequencing. Upon prenatal diagnosis, the fetus was found to carry two mutations. CONCLUSION: The combined next-generation sequencing and Sanger sequencing can reduce the time for diagnosis and identify subtypes of Fanconi anemia and the mutational sites, which has enabled reliable prenatal diagnosis of this disease.

Clinical features and ACADVL gene mutation spectrum analysis of 11 Chinese patients with very long chain acyl-CoA dehydrogenase deficiency / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the clinical and laboratory features of very long chain acyl-CoA dehydrogenase deficiency ( VLCADD ) and the correlations between its genotype and phenotype.</p><p><b>METHOD</b>Eleven patients diagnosed as VLCADD of Shanghai Jiaotong University School of Medicine seen from September 2006 to May 2014 were included. There were 9 boys and 2 girls, whose age was 2 d-17 years. Analysis was performed on clinical features, routine laboratory examination, and tandem mass spectrometry (MS-MS) , gas chromatography mass spectrometry (GC-MS) and genetic analysis were conducted.</p><p><b>RESULT</b>All cases had elevated levels of blood tetradecanoylcarnitine (C14:1) recognized as the characteristic biomarker for VLCADD. The eleven patients were classified into three groups: six cases in neonatal onset group, three in infancy onset group form patients and two in late onset group. Neonatal onset patients were characterized by hypoactivity, hypoglycemia shortly after birth. Infancy onset patients presented hepatomegaly and hypoglycemia in infancy. The two adolescent patients showed initial manifestations of exercise intolerance or rhabdomyolysis. Six of the eleven patients died at the age of 2-8 months, including four neonatal onset and two infant onset patients, with one or two null mutations. The other two neonatal onset patients were diagnosed since early birth through neonatal screening and their clinical manifestation are almost normal after treatments. Among 11 patients, seventeen different mutations in the ACADVL gene were identified, with a total mutation detection rate of 95.45% (21/22 alleles), including eleven reported mutations ( p. S22X, p. G43D, p. R511Q, p. W427X, p. A213T, p. C215R, p. G222R, p. R450H, p. R456H, c. 296-297delCA, c. 1605 + 1G > T) and six novel mutations (p. S72F, p. Q100X, p. M437T, p. D466Y, c. 1315delG insAC, IVS7 + 4 A > G). The p. R450H was the most frequent mutation identified in three alleles (13.63%, 3/22 alleles), followed by p. S22X and p. D466Y mutations which were detected in two alleles (9.09%, 2/22 alleles).</p><p><b>CONCLUSION</b>The ACADVL gene mutations were heterozygous in our patients. The mortality of neonatal onset form and infant onset form is much higher than the late onset form patients, suggesting a certain correlation between the genotype and phenotype was found. The earlier diagnosis and treatment of VLCADD are of vital importance for the improvement of the prognosis of the patients.</p>