[Application of next-generation sequencing technology for genetic diagnosis of Duchenne muscular dystrophy].

OBJECTIVE: To detect genetic causes of Duchenne muscular dystrophy (DMD). METHODS: Next-generation sequencing was used to detect 6 DMD patients in whom no exonic deletions were detected by multiplex PCR. Sanger sequencing and multiplex ligation-dependent probe amplification were used to confirm the results. RESULTS: One case was found to have deletions of exons 10 and 11, 1 had exons 16 and 17 duplication, 4 cases have 8 point mutations including c.2776C>T, c.5475delA, c.6391_6392delCA, IVS64+1G>A, c.2645A>G, c.5244G>A, c.7728T>C, c.8729A>T, c.8734A>G and c.8810G>A. The former 4 mutations are suspicious pathogenicity, the other 6 mutations are polymorphisms in population. Three novel mutations (IVS64+1G>A, c.6391_6392delCA (p.Q2131NfsX3) and p.Q926X (CAG>TAG) were not reported before. CONCLUSION: Next-generation sequencing technology is a useful tool for the detection of deletion, duplication and point mutation, which is valuable for clinical application.

[Analysis of GJB2 gene and mitochondrial DNA A1555G mutations in 16 families with non-syndromic hearing loss].

OBJECTIVE: To screen for genetic mutations in families featuring non-syndromic hearing loss. METHODS: Sixteen families with non-syndromic hearing loss were interviewed to identify medical histories by a questionnaire. Audiological and neurological examinations were conducted for all families. Coding regions of GJB2 and 12S rRNA genes were amplified and sequenced. RESULTS: Of the 17 patients with sensorineural hearing loss, 3 were homozygous mutation for GJB2 235 delC, 1 was 235 delC heterozygous mutation, 1 was 235 delC+299_300 delAT compound heterozygous mutation, and 6 were 79G>A+341G>A heterozygosis in cis mutation. No 1555A>G mutation of mitochondrial DNA (mtDNA) was found in the 16 families. CONCLUSION: The incidence of mtDNA 12S rRNA 1555A>G mutation in Jiangsu province may be lower than the average across China. Mutations of GJB2 genes may account for as much as 64.7% of non-syndromic hearing loss in this study. Screening for such mutations and genetic counseling may play an important role in the prevention of hereditary hearing loss.

[Genetic screening of mutation hot-spots for nonsyndromic hearing loss in southern Jiangsu province with SNaPshot].

OBJECTIVE: To investigate the mutation frequency in 7 mutation hot-spots of deafness gene in southern Jiangsu province and verify the performance of the SNaPshot technology platform, designed for genetic screening of non-syndromic hearing loss (NSHL) in Chinese. METHODS: One hundred and twenty-five NSHL patients were enrolled. Amplification of 235delC, 299-300delAT in GJB2 gene, IVS7-2A>G, 2168 A>G in SLC26A4 gene, and 1555A>G, 7445 A>G and 3243 A>G in mitochondrial DNA (mtDNA) was performed using multiplex polymerase chain reaction (PCR) technology. Afterwards, the sequence-specific probe interrogated each locus and labeled it at the 3' end using fluorescent dideoxynucleotide chemistry by the SNaPshot Multiplex Kit, the resulting products were then separated electrophoretically in ABI PRISM R 3130 Genetic Analyzer and analyzed in the presence of a fifth-dye-labeled size standard. Finally, the genotyping results were verified by direct sequencing or PCR-restriction fragment length polymorphism (PCR-RFLP). RESULTS: (1) The total mutation frequency for the 7 mutation hot-spots was 53.6%. The mutation frequency of 235delC was 24.0%, 299-300delAT was 5.6% in the GJB2 gene, IVS7-2A>G was 15.2%, 2168A>G was 3.2% in the SLC26A4 gene. The mutation frequency of 1555A>G and 7445 A>G in mtDNA was 4.8% and 0.8% respectively. The mutation 3243 A>G was not detected. (2) The SNaPshot results were consistent with that from direct sequencing or PCR-RFLP, and the specificity and sensitivity of detection were 100%. CONCLUSION: (1) More than half of the patients with deafness in southern Jiangsu province carry the mutations of the seven hot-spots. (2) The genetic screening technology platform based on SNaPshot can detect 7 mutations in one reaction, and is efficient and suitable for clinical practice.

[22q11 microdeletion test in patients with congenital heart defects by quantitative fluorescent PCR].

OBJECTIVE: To establish an assay for screening chromosome 22q11 microdeletion efficiently, and apply it for detecting del22q11 in patients with non-syndromic congenital heart defects (CHD). METHODS: Seventy nine patients with non-syndromic CHD and 84 normal controls were genotyped for 8 short tandem repeat (STR) markers located in 22q11 region, by using quantitative fluorescence polymerase chain reaction (QF-PCR). RESULTS: The average heterozygosity of the STR markers in patients and controls was 0.76 and 0.79, respectively. One patient with Tetralogy of Fallot (TOF) from the 79 CHD cases (1.3%) was found to have a deletion within chromosome 22q11.2, which was confirmed by multiplex ligation-dependent probe amplification (MLPA). CONCLUSION: The QF-PCR assay developed in this study was a reliable and an efficient alterative approach to screen for 22q11 microdeletion in clinical diagnosis and genetic counseling.

Correction to: miR-205-5p inhibits human endometriosis progression by targeting ANGPT2 in endometrial stromal cells.

An amendment to this paper has been published and can be accessed via the original article.

[Research on the illuminance characteristics of LED surguical luminaire].

The illuminance characteristics of LED (Lighting Emitting Diode) Surgical Luminaire is researched in this paper from the aspects of the LED Single-tube illumination analysis, LED arrays distribution and lighting design. The facula distribution characteristics of the LED prototype and multi-facet entirety reflection Surgical Luminaire is tested and compared according to the standards. The results of experimental show that LED prototype can fully meet the surgical illumination requirements, and the LED's own characteristics and further development will greatly broaden the space of medical lighting.

miR-205-5p inhibits human endometriosis progression by targeting ANGPT2 in endometrial stromal cells.

BACKGROUND: miRNA expression profiles in ectopic endometrium (EC) serving as pathophysiologic genetic fingerprints contribute to determining endometriosis progression; however, the underlying molecular mechanisms remain unknown. METHODS: miRNA microarray analysis was used to determine the expression profiling of EC fresh tissues. qRT-PCR was performed to screen miR-205-5p expression in EC tissues. The roles of miR-205-5p and its candidate target gene, angiopoietin-2 (ANGPT2), in endometriosis progression were confirmed on the basis of both in vitro and in vivo systems. miR-205-5p and ANGPT2 expression were measured by in situ hybridization and immunochemistry, and their clinical significance was statistically analysed. RESULTS: miR-205-5p was screened as a novel suppressor of endometriosis through primary ectopic endometrial stromal cell migration, invasion, and apoptosis assay in vitro, along with endometrial-like xenograft growth and apoptosis in vivo. In addition, ANGPT2 was identified as a direct target of miR-205-5p through bioinformatic target prediction and luciferase reporter assay. Re-expression and knockdown of ANGPT2 could respectively rescue and simulate the effects induced by miR-205-5p. Importantly, the miR-205-5p-ANGPT2 axis was found to activate the ERK/AKT pathway in endometriosis. Finally, miR-205-5p and ANGPT2 expression were closely correlated with the endometriosis severity. CONCLUSION: The newly identified miR-205-5p-ANGPT2-AKT/ERK axis illustrates the molecular mechanism of endometriosis progression and may represent a novel diagnostic biomarker and therapeutic target for disease treatment.

Levels of plasma des-gamma-carboxy protein C and prothrombin in patients with liver diseases.

AIM: To study the plasma des-gamma-carboxy protein C activity, antigen and prothrombin levels in patients with liver diseases and their clinical significance. METHODS: Plasma protein C activity (PC:C) was detected by chromogenic assay and antigen (PC:Ag) and des-gamma-carboxy protein C (DCPC) were detected by ELISA. Total prothrombin and unabsorbed prothrombin in plasma were detected by ecarin chromogenic assay. RESULTS: Compared with the control, the levels of PC:C and PC:Ag in patients with hepatocellular carcinoma (HCC) and liver cirrhosis (LC) were lower (PC:C: 104.65+/-23.0%, 62.50+/-24.89%, 56.75+/-20.14%, PC:Ag: 5.31+/-1.63 microg/mL, 2.28+/-1.15 microg/mL, 2.43+/-0.79 microg/mL, P<0.05). The levels of PC:Ag in patients with acute viral hepatitis (AVH) also was lower (2.98+/-0.91 microg/mL, P<0.01), but PC:C was close to the control (93.76+/-30.49%, P>0.05). The levels of DCPC in patients with HCC were remarkably higher (0.69+/-0.29 microg/mL, 1.18+/-0.63 microg/mL, 0.45+/-0.21 microg/mL, P<0.05) and its average was up to 50% of total PC:Ag. But those of DCPC in patients with AVH were not significantly different from the control. The levels of total prothrombin were lower in patients with LC, but higher in patients with HCC. The levels of unabsorbed prothrombin were predominantly higher than those of other groups. CONCLUSION: PC:C and PC:Ag in patients with liver diseases (except PC:C in AVH) were lower. The total prothrombin was lower in patients with LC. The higher level of unabsorbed prothrombin may be used as a scanning marker for HCC. DCPC may be used as a complementary marker in the diagnosis of HCC.

A patient with five chromosomal rearrangements and a 2q31.1 microdeletion.

BACKGROUND: Complex chromosomal rearrangements and chromosomal deletion and duplication syndromes are commonly associated with abnormal clinical phenotypes. The 2q31.1 microdeletion syndrome is a rare cytogenetic event that leads to limb and multi-internal organ anomalies. In this study we investigated the genetic basis of the physical and mental symptoms exhibited by a 4-year-old boy with a suspected 2q31.1 deletion. METHODS: Cytogenetic and molecular techniques including karyotyping, array-based comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH) and real-time PCR were used to identify the nature and extent of chromosome abnormalities in the patient. RESULTS: A 3.6Mb interstitial microdeletion of 2q31.1 was identified in association with complex balanced genomic structural rearrangements involving chromosomes 2, 3, 6, 15 and 18. The 2q31.1 deletion resulted in the loss of one copy of several known disease genes, including GAD1, DCAF17, SLC25A12 and ITGA6 associated with mental retardation and facial abnormalities and DLX1/DLX2 partially associated with limb abnormalities. Two additional genes, HOXD13 and CHN1, required for normal limb and eye development that map immediately distal to the 2q31.1 deletion had normal copy numbers, although CHN1 was found to express at a lower level in patient's lymphocytes. CONCLUSIONS: We speculated that the 2q31.1 deletion and/or translocation may have a positional effect which reduces expression of HOXD13 and CHN1 causing haplo-insufficiency, and in combination with the hemizygous expression of the disease genes at 2q31.1, provides a plausible explanation for the diverse clinical symptoms exhibited by the patient.

[Clinical detection of 22q11 microdeletion in the patients with congenital heart disease by multiplex ligation dependent probe amplification].

OBJECTIVE: To detect 22q11 microdeletion in the children and fetuses affected by congenital heart defects. METHOD: MLPA P250 kit was used to detect 22q11 microdeletion in 100 cases of sporadic congenital heart defects including 40 fetuses and 60 patients diagnosed by ultrasound. RESULT: Two cases from the fetuses and 1 case from the patients were found to have 22q11 microdeletion. CONCLUSION: Three cases had 22q11 microdeletion in the congenital heart defects.