Using whole exome sequencing and bioformatics in the molecular autopsy of a sudden unexplained death syndrome (SUDS) case.

Whole exome sequencing (WES) and bioinformatics analysis were used to investigate potential disease-causing gene mutations in a sudden unexplained death syndrome (SUDS) case after autopsy and pathology tests failed to suggest an obvious disease mechanism. Following whole exome sequencing, a 3-step bioinformatics filtering procedure was carried out to identify possible pathogenic genomic features. Single nucleotide variations (SNVs) were analyzed and ranked by likely mutation impact using various open online tools. After screening, we identified G643S as a putative causative heterozygous mutation in the KCNQ1 gene. This mutation has been reported in abnormalities consistent with SUDS, such as IKs in cardiac myocytes, a condition that predisposes for arrhythmias. Our work demonstrates the application of sequencing technology at the whole exome level for determining potential causes of an otherwise unexplained death.

[Molecular Genetic Analysis of One Sudden Unexplained Death in the Young by Whole Exome Sequencing].

OBJECTIVE: To find the mutation of disease-causing genes of sudden unexplained death syndrome (SUDS) in the young by whole exome sequencing in one case. METHODS: One SUDS case was found no obvious fatal pathological changes after conventional autopsy and pathological examination. The whole exome sequencing was performed with the Ion Torrent PGM™ System with hg19 as reference sequence for sequencing data. The functions of mutations were analyzed by PhyloP, PolyPhen2 and SIFT. A three-step bioinformatics filtering procedure was carried out to identify possible significative single nucleotide variation (SNV), which was missense mutation with allele frequency < 1% of myocardial cell. RESULTS: Four rare suspicious pathogenic SNV were identified. Combined with the analysis of conventional autopsy and pathological examination, the mutation MYOM2 (8_2054058_G/A) was assessed as high-risk deleterious mutation by PolyPhen2 and SIFT, respectively. CONCLUSION: Based on the second generation sequencing technology, analysis of whole exome sequencing can be a new method for the death cause investigation of SUDS. The gene MYOM2 is a new candidate SUDS pathogenic gene for mechanism research.

[A population genetic study of 22 autosomal loci of single nucleotide polymorphisms].

OBJECTIVE: To evaluate polymorphisms and forensic efficiency of 22 non-binary single nucleotide polymorphism (SNP) loci. METHODS: One hundred ethnic Han Chinese individuals were recruited from Dongguan, Guangdong. The 22 loci were genotyped with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). RESULTS: Nine loci were found with a single allele, 4 loci were found to be biallelic, whilst 9 loci were found to have 3 alleles. For 13 polymorphic loci, the combined discrimination power and power of exclusion were 0.999 98 and 0.9330, respectively. For the 9 non-biallelic loci, the combined discrimination power and power of exclusion were 0.9998 and 0.8956, respectively. For motherless cases, the combined power of exclusion was 0.6405 for 13 polymorphic SNPs and 0.6405 for 9 non-binary SNPs. CONCLUSION: Non-binary loci have a greater discrimination power and exclusion power per SNP.

[Estimation of postmortem interval by detecting thickness of cornea using ultrasonic method].

OBJECTIVE: To explore the postmortem changes of cornea thickness measured by ultrasonic pachymetry. METHODS: Eleven rabbits were randomly divided into two groups: one group with intact corneal epithelium and another group without intact corneal epithelium. In the later group, the corneal epithelium of the rabbit was scraped using mechanical elimination method. The corneal thickness was monitored continuously by ultrasonic pachymetry at several postmortem interval points in rabbits of the two groups. The changes of corneal thickness and postmortem interval were explored by relative regression analysis. RESULTS: The thickness of the cornea showed a strong non-linear correlation with the postmortem interval in the group with intact corneal epithelium. The group with intact corneal epithelium showed the correlation coefficient 0.922 and the group without intact corneal epithelium showed the correlation coefficient 0.822, respectively. CONCLUSION: The corneal thickness measured by ultrasonic pachymetry shows a potential value for estimating early postmortem interval. The intact corneal epithelium is a crucial factor for the measurement of cornea thickness by ultrasonic pachymetry.

[Effects of benzalkonium chloride on MUC1 in human conjunctival epithelial cells].

OBJECTIVE: To investigate the effects of benzalkonium chloride (BAC), a preservative used in many ophthalmic topical solutions, on mucin1 (MUC1) in human conjunctival epithelial cells in vitro. METHODS: Cultured epithelial cells obtained from human conjunctiva were exposed to medium containing BAC solutions at 0.0100%, 0.0050%, 0.0010%, 0.0005% and 0.0001% concentrations for a period of 15 min. Cells were examined after treatment and 6, 12, 24, 48 and 72 h later. The relative expression of the MUC1 mucin gene was determined by conventional reverse transcription-polymerase chain reaction (RT-PCR). Monoclonal antibody for MUC1 was used in Western blot analysis to detect MUC1. RESULTS: Cell exposure to 0.0100% and 0.0050% BAC decreased the expression of MUC1 at gene level between 12 and 72 h after treatment. Cells treated with 0.0010% and 0.0005% BAC decreased the expression of MUC1 between 24 and 48 h after treatment, recovered 72 h after treatment. At protein level, cells exposed to 0.0100% BAC decreased MUC1 between 24 and 72 h, 0.0050% BAC between 12 and 72 h, 0.0010% BAC 72 h later. CONCLUSIONS: These results suggest that BAC induces decreased expression of MUC1 at both gene and protein levels. The mode of BAC-induced decreased expression of MUC1 is dose-dependent.

[Exploration of lymphangiogenesis in alkali burned human cornea].

OBJECTIVE: To explore the lymphangiogenesis process in alkali burned human cornea and to discuss factors modulating this process. METHODS: It was a retrospective case series study. Twenty-two cases (22 eyes) of hospitalized patients suffering from alkali burned cornea and requiring keratoplasty from January to December 2005 were analyzed retrospectively. Before surgery, injury time (IT) and injury degree (ID) were recorded. Furthermore, inflammation index (II) and relative area of new blood vessels (BVA) were measured. Cornea specimens were assessed for lymphatic vessel counting (LVC) and blood vessel counting (BVC) via immunohistochemical staining and transmission electronic microscopy. Meanwhile, HE staining was also performed to observe infiltration of polymorphonuclear (PMN) leucocytes in corneal tissues. Student t-test, Pearson correlation test and Stepwise regression analysis were used to investigate the influencing factors. RESULTS: In these 22 cases, IT was (57.62 +/- 31.72) months; ID was (12.00 +/- 2.76) scores; II was (2.32 +/- 2.63) scores; BVA was (29.79% +/- 18.61%); BVC was (14.45 +/- 9.29) units; LVC was (2.73 +/- 4.57) units and PMN was (13.45 +/- 13.09) units. In 7 patients with IT more than 64 months (accounted for 32% of 22 cases), lymphangiogenesis [(8.6 +/- 3.8) units] and hemangiogenesis [(22.3 +/- 11.1) units] were both present. In these 7 patients, the whole number of LVC was 60 units, constituting 16% of all vessels (BVC+LVC = 378 units). The correlation coefficient of LVC with IT, ID, BVA, PMN and II was -0.673, 0.604, 0.755, 0.806 and 0.873, respectively. P value of all these correlations was less than 0.05. Further regression analysis revealed that LVC could be approximately calculated from II and BVA multiplying certain constant coefficients separately (resulting in lymphatic index, LI). Lymphatic vessels with characteristic ultrastructures and inflammatory cells were identified by transmission electronic microscopy. CONCLUSIONS: Lymphatic vessels exist in part of alkali burned human corneas and may be estimated through II and BVA indirectly. Lymphatic index may be a convenient and useful clinical index for evaluating lymphangiogenesis in corneal alkali burn.

Pericytes are correlated with the permeability of rat corneal neovascular vessels induced by alkali burn.

BACKGROUND: Corneal neovascular leakage can lead to edema and secondary scarring. Previous studies have shown that pericytes play a key role in maturation of angiogenesis. The present studies investigate the relationship between vascular permeability and pericyte coverage of endothelial cells in rat corneal neovascular induced by alkali burns. METHODS: Corneal neovascular vessels induced by alkali burns was performed in Sprague-Dawley rats. Corneas were excised on 1, 2, 3, 5, 7 and 10 days after cauterization. The vascular permeability rate was measured by the Evans blue method. The microvessel pericyte coverage index (MPI) was applied to quantify the pericyte coverage through double immunofluorescent staining of frozen sections of corneas with CD31 as the endothelial and alpha-smooth muscle actin (alpha-SMA) as the pericyte markers. The correlation between permeability rate and MPI was analyzed. Pericyte coverage was confirmed ultrastructually using transmission electron microscopy. RESULTS: The vascular permeability rate was (1.14 +/- 0.17), (0.24 +/- 0.08), (0.29 +/- 0.16), (0.14 +/- 0.10), (0.09 +/- 0.06) and (0.05 +/- 0.04) microg x ml(-1) x mm(-2) respectively on 1, 2, 3, 5, 7 and 10 days after cauterization. The MPI was 0, 16.07%, 11.95%, 43.84%, 73.97% and 86.21% respectively at the above mentioned time points. The correlation coefficient between MPI and the permeability rate was -0.943 (P = 0.005). CONCLUSIONS: Pericyte recruitment was significantly correlated with the permeability of corneal neovascularization induced by alkali burns in rats. Therapeutic strategies aiming at anti-leakage should be most effective if they promote pericytes proliferation in the course of corneal neovascularization.

[Quantification of oxygen-induced retinopathy in the mouse].

PURPOSE: To establish a quantifying model of retinal neovascularization suitable for examining pathogenesis and therapeutic intervention for the retinal neovascularization. METHODS: Sixty 7-day-old C57BL/6J mice were divided into oxygen-induced retinopathy group and control group. In oxygen-induced retinopathy group, 32 mice were exposed to (75 +/- 2)% oxygen for 5 days and then to room air; in control group, 32 mice were raised in room air. The retinal frozen sections were stained with griffonia simplicifolia lectin B4 (GSA) which selectively stained vascular cells; or the retinal preparation perfused with fluorescein-dextran were used to test the areas of the retinal neovascularization. RESULTS: After 5 days of exposure to hyperoxia at postnatal day 12 (P12), the larger central radial vessels became tortuous and constricted and central perfusion became obviously decreased. After returning to room air for 2 days at P14, neovascularization was seen. This response was maximal at P17. CONCLUSION: The reproducible and quantifiable mouse model of retinal neovascularization is useful for the study of pathogenesis of retinal neovascularization and therapeutic intervention.

In vivo and in vitro inhibitory effect of amniotic extraction on neovascularization.

PURPOSE: To prepare amniotic extraction (AE) and to test its antiangiogenic effect in vivo and in vitro. METHODS: AE was prepared and diluted to 50, 100, and 200 microg/mL concentrations. Alkali burn-induced corneal neovascularization (NV) was produced and topically treated with different concentrations of AE or 0.1% dexamethasone for 7 days. Normal saline was used as a control. Corneal NV was visualized by heart perfusion of Chinese ink and quantified as the percentage of corneal NV area to the whole corneal area. Human umbilical vein endothelial cells (HUVECs) were primarily cultured. The effects of AE on proliferation and tube formation of HUVECs were tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and in vitro angiogenesis assay. Pigment epithelium-derived factor (PEDF) in AE was detected by Western blot. RESULTS: Relative corneal NV area in the control group was 56.6% +/- 9.9%, which was significantly reduced by 50 microg/mL AE (47.6% +/- 6.9%; P = 0.043) and 200 microg/mL AE (34.3% +/- 7.8%; P < 0.001) and by 0.1% dexamethasone (21.1% +/- 1.8%; P < 0.001). HUVEC cell proliferation was significantly decreased after treatment with AE at concentrations of 50 and 100 microg/mL compared with control (P = 0.036 and 0.001, respectively). The tube formation was significantly suppressed by 100 microg/mL AE (70.03% +/- 4.35%) compared with control (100% +/- 4.84%; P = 0.002). No expression of PEDF was detected in AE. CONCLUSION: AE inhibits NV induced by alkali burn. This effect may be elicited at least in part through the inhibiting activity of blood vessel endothelial cells and is not associated with PEDF.

Rapid detection of six common Chinese G6PD mutations by MALDI-TOF MS.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked hereditary enzymopathy. We describe here the techniques based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and multiprimer extension (multi-PEX) to detect the most common Chinese G6PD mutations, which are the single-point mutations G-->T at nt 1376, G-->A at nt 1388, A-->G at nt 95, G-->T at nt 392, C-->T at nt 1024, and C-->T at nt 1311. Fifteen samples were genotyped using this method coupled with direct sequencing, after identification of G6PD mutations by ARMS. In this study, we identified a mutation G-->T at nt 1376, which had been G-->A at nt 1388 using ARMS, while the result of sequencing corresponds with ours. This indicates the reliability of this method. Furthermore, since it can scan six common Chinese G6PD mutations simultaneously in one mass spectrum, this approach could be used to fast diagnose these G6PD mutations accurately in large-scale analysis.

[Studying the polymorphism at DYF155S1 locus in Chinese Han population by MVR-PCR marked with fluorescence].

The simple and useful genotyping methods of DYF155S1 locus by silver staining and fluorescence detection have been established. The blood samples from 155 unrelated males in Chinese Han population were typed by these two methods respectively and the same results were obtained. Among the 155 samples 66 alleles were found, out of them 38 were observed once only. The most frequent alleles named 18 or 22, which frequency was 0.065, the size of their first DNA fragments was 180 bp and the number of fragments was 16 or 17. The gene diversity (h) was 0.9789. Out of the 155 samples, 25 samples had one or two bands lost (null repeat) among the successive bands. It was demonstrated by sequencing that the position of the lost bands corresponded to type 3 repeats. Our results indicated that the method, which revealed the 5' end diversity of DYF155S1 locus, was a technique that could obtain the most Y-specific polymorphic information only by one PCR reaction. The allele frequencies of DYF155S1 locus in Chinese Han population provided the basic data for the study of population genetics and forensic practices.