[Screening of the SETX gene in sporadic amyotrophic lateral sclerosis patients of Chinese origin].

Objective: To investigate all coding regions of amyotrophic lateral sclerosis (ALS)-related gene Senataxin (SETX) in sporadic amyotrophic lateral sclerosis patients of Chinese origin. Methods: From January 2010 to December 2014, the peripheral venous blood samples and clinical data were collected from 311 patients with sporadic amyotrophic lateral sclerosis (SALS) and 311 healthy controls who were of Chinese ancestry from the Department of Neurology, Chinese PLA General Hospital.Genomic DNA was extracted from peripheral venous blood of all participants using standard methods. The coding regions of SETX were amplified by polymerase chain reaction (PCR) and screened for mutations using next-generation sequencing technology. The online software SIFT and PolyPhen-2 were used to analyze the conservation of an altered amino acid and predict the potential pathogenicity of identified mutations. The SPSS 22.0 software was used to analyze the clinical feature of all participants. Results: Tenkinds of rare and one novel nonsynonymous mutations were identified and were absent in 311 controls. Twelve (3.86%) patients carried one SETX gene mutation. Five (1.61%) out of above-mentioned 12 patients carried highly pathogenic mutations including p. Pro1868Leu (c.5603G>A), p. Pro1331Leu (c.3992G>A), p. Glu756Val (c.2267T>A), p. Leu564Val (c.1690A>C), and p. Asn144Ser (c.431T>C). Patients carried SETX mutations were not different from other patients in onset age. Conclusion: Mutations in SETX are likely to be a pathogenesis for Chinese sporadic amyotrophic lateral sclerosis.

[Expression of long noncoding RNA STCAT3 in gastric cancer tissues and its effect on malignant phenotype of gastric cancer cells].

Objective: To detect the expression of long noncoding RNA(lncRNA)stomach cancer-associated transcript-3(STCAT3) in gastric cancer tissues, adjacent tissues, human gastric cancer cell lines and normal gastric epithelial cell lines, and to investigate the relationship between STCAT3 expression and clinicopathological features and malignant phenotype of gastric cancer. Methods: Quantitative fluorescent real-time polymerase chain reaction (qRT-PCR) was applied to detect the lncRNA STCAT3 expression levels in gastric cancer tissues, paired adjacent non-tumorous tissues, in order to explore the relationship between STCAT3 expression and clinicopathological features of gastric cancer. lncRNA STCAT3 low-expressing and high-expressing gastric cancer cell lines were transfected with expression plasmid to simulate gain-of-function, or interference plasmid to achieve loss-of-function. Cell proliferation was measured with CCK-8 and colony formation assay, cell migration with scratch assay, and cell invasion with Transwell migration assay. human gastric tumor were also transplanted to nude mice to detect the effect of lncRNA STCAT3 on tumorigenesis. Results: The expression of lncRNA STCAT3 was generally up-regulated in gastric cancer tissues compared with the adjacent tissues(12.55±0.16 vs 6.52±0.14), with median expression level in gastric cancer tissues being 6.03 higher (P<0.05). Meanwhile, the expression level of lncRNA STCAT3 in gastric cancer tissues was not correlated with age or gender (both P>0.05), while positively correlated with TNM stage (P<0.05). Interference of lncRNA STCAT3 expression in BGC-823 cells was found associated with significantly suppressed colony formation, proliferation, invasion, and migration (all P<0.05). Over-expression of lncRNA STCAT3 in AGS cells were also founded could promote the gastric cancer cells' proliferation, colony formation, migration, and invasion (all P<0.05). Conclusions: lncRNA STCAT3 may participate in the proliferation and invasion of gastric cancer cells, indicating that dysregulation of STCAT3 expression may play a role in occurrence and development of gastric cancer. lncRNA STCAT3 has the potential to be the biomarker of gastric cancer progression and target in treatment. The underlying mechanism is yet to be further studied.

[Mutation screening for the causative gene in a four-generation Chinese pedigree with progressive cardiac conduction defect].

OBJECTIVE: To define the potential causative gene mutation in a Chinese pedigree with progressive cardiac conduction defect (PCCD). METHODS: Sanger sequencing was performed to define potential causative gene mutation in a four-generation family with 68 members including seven PCCD patients (5 male) from 2010 to 2015.No causative gene was detected by screening known candidate genes related to PCCD including SCN5A, NKX2.5 and LMNA.High-throughput sequencing technology on exon-enriched DNA was then used to search the causative genes in 2 patients and one normal family member. RESULTS: Eight new non-synonymous single nucleotide variants including AQP7 gene (exon5: c.T343C: p.Y115H), CACNA1B gene (NM_001243812: exon19: c.A2986G: p.T996A), CATSPERB gene (exon27: c.C3254G: p.P1085R), CLCA2 gene (exon11: c.G1725T: p.W575C), CLCA3P gene (ncRNA_intronic), MYLK-AS1 gene (ncRNA_intronic), TTN gene (ncRNA_UTR3), LMNA gene (LMNA: NM_170708: exon5: c.C922T: p.Q308X) were identified by comparing and filtering the results with known public databases.Then, more detailed biological analysis on these 8 genes was conducted.Traditional Sanger sequencing validated the exome sequencing results, and found that the mutation c. 1725Gï¹¥T in gene CLCA2 segregated with the phenotype of this PCCD pedigree.The mutation c. 1725Gï¹¥T in gene CLCA2 was thus be considered as the causative PCCD gene in this pedigree from the perspective of genetics and genomics. CONCLUSION: The heterozygote mutation c. 1725Gï¹¥T in gene CLCA2 might be causative gene in this PCCD pedigree.This finding adds new gene mutation variant responsible for PCCD.

Regulating effect of MMP-9 and TIMP-1 in pituitary adenoma invasion.

Pituitary adenomas can cause endocrine disorder and organ damage, with some aggressive ones leading to a high postoperative recurrence rate. The occurrence and development of these type of tumors is closely related with matrix metalloproteinases (MMPs) and endogenous specific tissue inhibitor of MMPs (TIMPs). In this study, the relationship between pituitary adenoma invasion and the changes in MMP-8 and TIMP-1 expressions is analyzed. Specimens from sixty patients with pituitary adenoma were collected in our hospital after surgery, including thirty cases of invasive pituitary adenomas and thirty cases of noninvasive pituitary adenomas. Western blotting and real-time PCR were used to detect MMP-8/TIMP-1 protein and mRNA levels, respectively, in the two types of pituitary adenomas, while ELISA was used to detect both compounds' levels in the patient's serum. Compared with noninvasive pituitary adenomas, MMP-8 was significantly overexpressed in invasive pituitary adenomas, while TIMP-1 was obviously lower (P < 0.05 for both). Moreover, MMP-8 mRNA expression in invasive pituitary adenomas was significantly higher than in noninvasive pituitary adenomas, while TIMP-1 mRNA expression was markedly lower (P < 0.05 for both). Finally, MMP-8 expression in the serum is upregulated in patients with invasive pituitary adenomas relative to the noninvasive ones, and the expression of TIMP-1 significantly reduced (P < 0.05 for both). These results show that increased MMP-8 and decreased TIMP-1 expressions are closely related to the invasive pituitary adenoma, and can be helpful for the evaluation.

Maternal depression is associated with DNA methylation changes in cord blood T lymphocytes and adult hippocampi.

Depression affects 10-15% of pregnant women and has been associated with preterm delivery and later developmental, behavioural and learning disabilities. We tested the hypothesis that maternal depression is associated with DNA methylation alterations in maternal T lymphocytes, neonatal cord blood T lymphocytes and adult offspring hippocampi. Genome-wide DNA methylation of CD3+ T lymphocytes isolated from 38 antepartum maternal and 44 neonatal cord blood samples were analyzed using Illumina Methylation 450 K microarrays. Previously obtained methylation data sets using methylated DNA immunoprecipitation and array-hybridization of 62 postmortem hippocampal samples of adult males were re-analyzed to test associations with history of maternal depression. We found 145 (false discovery rate (FDR) q<0.05) and 2520 (FDR q<0.1) differentially methylated CG-sites in cord blood T lymphocytes of neonates from the maternal depression group as compared with the control group. However, no significant DNA methylation differences were detected in the antepartum maternal T lymphocytes of our preliminary data set. We also detected 294 differentially methylated probes (FDR q<0.1) in hippocampal samples associated with history of maternal depression. We observed a significant overlap (P=0.002) of 33 genes with changes in DNA methylation in T lymphocytes of neonates and brains of adult offspring. Many of these genes are involved in immune system functions. Our results show that DNA methylation changes in offspring associated with maternal depression are detectable at birth in the immune system and persist to adulthood in the brain. This is consistent with the hypothesis that system-wide epigenetic changes are involved in life-long responses to maternal depression in the offspring.

Nonredundant roles of the mPer1 and mPer2 genes in the mammalian circadian clock.

Mice carrying a null mutation in the Period 1 (mPer1) gene were generated using embryonic stem cell technology. Homozygous mPer1 mutants display a shorter circadian period with reduced precision and stability. Mice deficient in both mPer1 and mPer2 do not express circadian rhythms. While mPER2 regulates clock gene expression at the transcriptional level, mPER1 is dispensable for the rhythmic RNA expression of mPer1 and mPer2 and may instead regulate mPER2 at a posttranscriptional level. Studies of clock-controlled genes (CCGs) reveal a complex pattern of regulation by mPER1 and mPER2, suggesting independent controls by the two proteins over some output pathways. Genes encoding key enzymes in heme biosynthesis are under circadian control and are regulated by mPER1 and mPER2. Together, our studies show that mPER1 and mPER2 have distinct and complementary roles in the mouse clock mechanism.

MPer1 and mper2 are essential for normal resetting of the circadian clock.

Mammalian Per1 and Per2 genes are involved in the mechanism of the circadian clock and are inducible by light. A light pulse can evoke a change in the onset of wheel-running activity in mice by shifting the onset of activity to earlier times (phase advance) or later times (phase delays) thereby advancing or delaying the clock (clock resetting). To assess the role of mouse Per (mPer) genes in circadian clock resetting, mice carrying mutant mPer1 or mPer2 genes were tested for responses to a light pulse at ZT 14 and ZT 22, respectively. The authors found that mPer1 mutants did not advance and mPer2 mutants did not delay the clock. They conclude that the mammalian Per genes are not only light-responsive components of the circadian oscillator but also are involved in resetting of the circadian clock.

Autoimmune hypogonadism as part of an autoimmune polyglandular syndrome.

The most compelling case for autoimmune mediated hypogonadism occurs when ovarian failure is part of an autoimmune polyglandular syndrome (APS). In patients with the rare, recessively inherited type 1 APS (APS-1), characterized by the triad of chronic mucocutaneous moniliasis, hypoparathyroidism, and Addison's disease, primary amenorrhea (elevated pituitary gonadotropins) or oligomenorrhea and infertility are constant features. Ovarian failure is associated with autoantibodies to steroid hormone secreting cells in the adrenal cortex, Leydig cells of the testes, granulosa/thecal cells of the Graffian follicles, corpus luteum, and the syncytiotrophoblast of the placenta. These autoantibodies react with 3 P450 enzymes involved with steroidogenesis, namely, 21-hydroxylase (adrenal specific), 17 alpha-hydroxylase, and the side chain cleavage enzyme. Recently the 14 exon, APS-1 (autoimmune regulator or AIRE) gene has been cloned (chr. 21p22.3), and multiple mutants discovered. Parents who are obligatory heterozygotes for a single mutant gene lack clinical features of APS-1. They also do not develop APS-1 autoantibodies. Thus, hypogonadal patients without features of APS-1 are unlikely to have AIRE gene mutations. In the more common APS-2/3, characterized by combinations of autoimmune thyroid disease, immune mediated type 1 diabetes, vitiligo, pernicious anemia, and Addison's disease (type 2, not type 3), ovarian disease may be seen. In primary hypogonadism outside of the context of an APS, these autoantibodies are rare.

Limited genetic susceptibility to severe Graves' ophthalmopathy: no role for CTLA-4 but evidence for an environmental etiology.

Graves' disease (GD) is an autoimmune thyroid disease (AITD) characterized by hyperthyroidism and by the occurrence of a distinctive ophthalmopathy (orbitopathy), which presents with varying degrees of severity. Graves' disease clusters in families but the importance of heredity in the pathogenesis of the associated ophthalmopathy is unclear. We have studied the family history of 114 consecutive, ethnically mixed patients with severe Graves' ophthalmopathy (GO). Patients were selected by unambiguous single ascertainment. Seventy-seven percent of patients were female and 59% smoked. The mean age at onset of their GD was 43 years (range 17-78 years). Forty-one patients (36%) had a family history of AITD, defined as a first- and/or a second-degree relative affected with either Graves' disease (GD) or Hashimoto's thyroiditis (HT). The segregation ratio for AITD in nuclear families in our ascertained Graves' ophthalmopathy families was 0.07 (0.12 in Caucasians only). Hence, the higher prevalence of AITD among relatives of Graves' ophthalmopathy patients agreed with the known genetic predisposition to AITD and this predisposition was stronger in women than in men. However, only 3 of the 114 patients had a family history of severe Graves' ophthalmopathy (all second-degree relatives) and the segregation ratio for GO was 0. These data did not support a major role for familial factors in the development of severe Graves' ophthalmopathy distinct from Graves' disease itself. In addition, we tested 4 candidate genes, human leukocyte antigen (HLA), tumor necrosis factor-beta (TNF-beta), CTLA-4 and the thyrotropin receptor (TSHR), for association with Graves' ophthalmopathy. These were negative except for the HLA and CTLA-4 genes, which were found to be weakly associated with GO giving similar relative risk (RR) as in GD patients without ophthalmopathy. These data suggested that environmental factors, rather than major genes, were likely to predispose certain individuals with AITD to severe Graves' ophthalmopathy. Smoking remains one example of such potential external insults.

The mPer2 gene encodes a functional component of the mammalian circadian clock.

Circadian rhythms are driven by endogenous biological clocks that regulate many biochemical, physiological and behavioural processes in a wide range of life forms. In mammals, there is a master circadian clock in the suprachiasmatic nucleus of the anterior hypothalamus. Three putative mammalian homologues (mPer1, mPer2 and mPer3) of the Drosophila circadian clock gene period (per) have been identified. The mPer genes share a conserved PAS domain (a dimerization domain found in Per, Arnt and Sim) and show a circadian expression pattern in the suprachiasmatic nucleus. To assess the in vivo function of mPer2, we generated and characterized a deletion mutation in the PAS domain of the mouse mPer2 gene. Here we show that mice homozygous for this mutation display a shorter circadian period followed by a loss of circadian rhythmicity in constant darkness. The mutation also diminishes the oscillating expression of both mPer1 and mPer2 in the suprachiasmatic nucleus, indicating that mPer2 may regulate mPer1 in vivo. These data provide evidence that an mPer gene functions in the circadian clock, and define mPer2 as a component of the mammalian circadian oscillator.

RIGUI, a putative mammalian ortholog of the Drosophila period gene.

The molecular components of mammalian circadian clocks are elusive. We have isolated a human gene termed RIGUI that encodes a bHLH/PAS protein 44% homologous to Drosophila period. The highly conserved mouse homolog (m-rigui) is expressed in a circadian pattern in the suprachiasmatic nucleus (SCN), the master regulator of circadian clocks in mammals. Circadian expression in the SCN continues in constant darkness, and a shift in the light/dark cycle evokes a proportional shift of m-rigui expression in the SCN. m-rigui transcripts also appear in a periodic pattern in Purkinje neurons, pars tuberalis, and retina, but with a timing of oscillation different from that seen in the SCN. Sequence homology and circadian patterns of expression suggest that RIGUI is a mammalian ortholog of the Drosophila period gene, raising the possibility that a regulator of circadian clocks is conserved.

Isolation of novel genes from the CMT1A duplication/HNPP deletion critical region in 17p11.2-p12.

Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with a 1.5-Mb tandem DNA duplication in chromosome 17p11.2-p12, while hereditary neuropathy with liability to pressure palsies (HNPP) is associated with a 1.5-Mb deletion at this locus. The 1.5-Mb CMT1A monomer unit duplicated in CMT1A and deleted in HNPP is flanked by low-copy repeats termed CMT1A-REPs. Both diseases appear to be caused by an altered copy number of the peripheral myelin protein 22 gene (PMP22), which lies within the critical region. To identify additional genes rapidly, we used a cosmid contig of this region and reciprocal probing of arrayed chromosome 17-specific cosmid and cDNA libraries. Three cDNA clones were identified within the CMT1A duplication/HNPP deletion region and one just proximal to the critical region. The cDNA for human heme A:farnesyltransferase (COX10) mapped 10 kb centromeric to the distal CMT1A-REP. The other two cDNA clones from within the critical interval mapped to cosmid 126D1 at the mfd41 (D17S261) DNA marker, and their conceptual translation showed homology to 60S ribosomal protein L9 (RPL9) and chromosomal protein RMSA-1 (RMSA-1). A gene that is homologous to human peroxisome proliferator activated receptor alpha (hPPARA) was identified near the proximal CMT1A-REP.

[Thin skin flap in the repair of defect of soft tissue of hand].

To meet the special need of function and cosmetics, thin skin flap was used in repair of skin defect of hand. It was reported that 37 cases with skin defect of hand had been treated since 1992. Eight kinds of flaps were used, including sub-abdominal flap (in 8 cases), paraumbilical flap (in 8 cases), intercostal flap (in 5 cases), subclavicular flap (in 3 cases), thoracoacromial flap (in 3 cases), trigonum clavopectoral flap (in 4 cases), thenal flap (in 2 cases) and pedicled flap carrying superficial iliac circumflex artery (in 4 cases). All of defects were treated by one-stage operation. The size of the flaps used ranged from the minimum of 1.2 cm x 2.5 cm to the maximum of 10 cm x 16 cm. All the flaps were survived. The function and appearance of the hands were satisfactory. It was noted that thin skin flap had developed from conventional pedicled flap and subcorium vascular-net flap, but it was easy to perform and had a high rate of success. The principle in choosing the flap was also discussed. It was suggested that more attention should be concentrated to solve the problems encountered in the use of thin skin flap in the repair of skin defect.

A differential response of two putative mammalian circadian regulators, mper1 and mper2, to light.

A mouse gene, mper1, having all the properties expected of a circadian clock gene, was reported recently. This gene is expressed in a circadian pattern in the suprachiasmatic nucleus (SCN). mper1 maintains this pattern of circadian expression in constant darkness and can be entrained to a new light/dark cycle. Here we report the isolation of a second mammalian gene, mper2, which also has these properties and greater homology to Drosophila period. Expression of mper1 and mper2 is overlapping but asynchronous by 4 hr. mper1, unlike period and mper2, is expressed rapidly after exposure to light at CT22. It appears that mper1 is the pacemaker component which responds to light and thus mediates photic entrainment.

Isolation, mapping, and genomic structure of an X-linked gene for a subunit of human mitochondrial complex I.

We report here the isolation, mapping, and genomic organization of the human NDUFA1 gene, which is a component of NADH:ubiquinone oxidoreductase (complex I). The NDUFA1 cDNA clone and associated genomic cosmid clones were isolated by reciprocal probing of an arrayed human heart cDNA library with a X-chromosome cosmid library and were mapped to Xq24. The NDUFA1 gene, which is highly expressed in human cardiac and skeletal muscle, has an open reading frame of 70 amino acids and shows 80% homology to the bovine MWFE subunit of complex I. By primer extension, the major and minor transcription initiation sites were identified, 99 and 141 nucleotides upstream of the translation initiator ATG, respectively. The NDUFA1 gene is composed of 3 exons and spans about 5.0 kb of genomic DNA. The 5' region of the NDUFA1 gene (approximately 450-bp fragment) lacks conventional TATA and CAAT boxes, but it contains several potential binding sites for transcription factors including SP-1, AP-2, NF1, NRF2-like, APRRE, CRE, MyoD1, CArG, MEF-2, and BRE.