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Background Nuclear-derived cell-free DNA (cfDNA) molecules in blood plasma are nonrandomly fragmented, bearing a wealth of information related to tissues of origin. DNASE1L3 (deoxyribonuclease 1 like 3) is an important player in shaping the fragmentation of nuclear-derived cfDNA molecules, preferentially generating molecules with 5 CC dinucleotide termini (i.e., 5 CC-end motif). However, the fragment end properties of microbial cfDNA and its clinical implication remain to be explored. Methods We performed end motif analysis on microbial cfDNA fragments in plasma samples from patients with sepsis. A sequence context-based normalization method was used to minimize the potential biases for end motif analysis. Results The end motif profiles of microbial cfDNA appeared to resemble that of nuclear cfDNA (Spearman correlation coefficient: 0.82, P value 0.001). The CC-end motif was the most preferred end motif in microbial cfDNA, suggesting that DNASE1L3 might also play a role in the fragmentation of microbe-derived cfDNA in plasma. Of note, differential end motifs were present between microbial cfDNA originating from infection-causing pathogens (enriched at the CC-end) and contaminating microbial DNA potentially derived from reagents or the environment (nearly random). The use of fragment end signatures allowed differentiation between confirmed pathogens and contaminating microbes, with an area under the receiver operating characteristic curve of 0.99. The performance appeared to be superior to conventional analysis based on microbial cfDNA abundance alone. Conclusions The use of fragmentomic features could facilitate the differentiation of underlying contaminating microbes from true pathogens in sepsis. This work demonstrates the potential usefulness of microbial cfDNA fragmentomics in metagenomics analysis.

Single-stranded ends of double-stranded DNA (jagged ends) are more abundant in urinary DNA than in plasma DNA. However, the lengths of jagged ends in urinary DNA remained undetermined, as a previous method used for urinary DNA jagged end sequencing analysis (Jag-seq) relied on unmethylation at CpG sites, limiting the resolution. Here, we performed high-resolution Jag-seq analysis using methylation at non-CpG cytosine sites, allowing determination of exact length of jagged ends. The urinary DNA bore longer jagged ends (~26-nt) than plasma DNA (~17-nt). The jagged end length distribution displayed 10-nt periodicities in urinary DNA, which were much less observable in plasma DNA. Amplitude of the 10-nt periodicities increased in patients with renal cell carcinoma. Heparin treatment of urine diminished the 10-nt periodicities. The urinary DNA jagged ends often extended into nucleosomal cores, suggesting potential interactions with histones. This study has thus advanced our knowledge of jagged ends in urine DNA.

Objective Cell‐free DNA (cfDNA) fragments in maternal plasma contain DNA damage and may negatively impact the sensitivity of noninvasive prenatal testing (NIPT). However, some of these DNA damages are potentially reparable. We aimed to recover these damaged cfDNA molecules using PreCR DNA repair mix. Methods cfDNA was extracted from 20 maternal plasma samples and was repaired and sequenced by the Illumina platform. Size profiles and fetal DNA fraction changes of repaired samples were characterized. Targeted sequencing of chromosome Y sequences was used to enrich fetal cfDNA molecules following repair. Single‐molecule real‐time (SMRT) sequencing platform was employed to characterize long (>250 bp) cfDNA molecules. NIPT of five trisomy 21 samples was performed. Results Size profiles of repaired libraries were altered, with significantly increased long (>250 bp) cfDNA molecules. Single nucleotide polymorphism (SNP)‐based analyses showed that both fetal‐ and maternal‐derived cfDNA molecules were enriched by the repair. Fetal DNA fractions in maternal plasma showed a small but consistent (4.8%) increase, which were contributed by a higher increment of long fetal cfDNA molecules. z‐score values were improved in NIPT of all trisomy 21 samples. Conclusion Plasma DNA repair recovers and enriches long cfDNA molecules of both fetal and maternal origins in maternal plasma., What is already known about this topic? Most of the cell‐free DNA (cfDNA) fragments in maternal plasma have sizes less than 200 bp, with fetal molecules being shorter than maternal ones.DNA damages exist in cfDNA, particularly single‐strand nicks.Occasional no call for noninvasive prenatal testing (NIPT) can be caused by insufficient fetal DNA fraction. What does this study add? Repair of cfDNA by PreCR repair mix can recover a subset of long (>250 bp) cfDNA molecules.Both fetal and maternal long cfDNA are enriched by PreCR repair treatment.Mild but consistent increments in fetal DNA fractions after PreCR repair, which are contributed by higher enrichment of long fetal cfDNA molecules.PreCR repair treatment improves NIPT of trisomy 21 by elevating z scores resulting in better discrimination of aneuploid from euploid samples.

International audience; Plasma DNA fragmentomics is an emerging area in cell-free DNA diagnostics and research. In murine models, it has been shown that the extracellular DNase, DNASE1L3, plays a role in the fragmentation of plasma DNA. In humans, DNASE1L3 deficiency causes familial monogenic systemic lupus erythematosus with childhood onset and anti-dsDNA reactivity. In this study, we found that human patients with DNASE1L3 disease-associated gene variations showed aberrations in size and a reduction of a ''CC'' end motif of plasma DNA. Furthermore, we demonstrated that DNA from DNASE1L3-digested cell nuclei showed a median length of 153 bp with CC motif frequencies resembling plasma DNA from healthy individuals. Adeno-associated virus-based transduction of Dnase1l3 into Dnase1l3-deficient mice restored the end motif profiles to those seen in the plasma DNA of wild-type mice. Our findings demonstrate that DNASE1L3 is an important player in the fragmentation of plasma DNA, which appears to act in a cell-extrinsic manner to regulate plasma DNA size and motif frequency.

Urinary cell-free (cf) DNA holds great potential as a completely noninvasive form of liquid biopsy. Knowledge of the composition of cfDNA by tissue of origin is useful for guiding its clinical uses. We conducted a global survey of urinary cfDNA composition using genomewide bisulfite sequencing. While previous studies focused on detecting cfDNA from a single source at a time, genomewide tissue specific methylation signatures allow us to simultaneously deduce the proportional contribution from each contributing tissue. The proportional contributions derived from methylation deconvolution are highly correlated with those calculated using allograft-derived donor-specific genetic markers in the urine of hematopoetic stem cell and renal transplant recipients. We found a large variation of proportional contributions from different tissues. We then assessed if cfDNA undergoes time-dependent fragmentation in urine by conducting in vitro incubation experiments. In vitro incubation at 37°C showed that urinary cfDNA concentration decreased under first order kinetics with a half-life of 2.6 to 5.1h. This is reflected in parallel by a decrease in the proportion of long fragments and increase in amplitude of 10bp periodicity seen in the cfDNA size profile. This global survey of urinary cfDNA has deepened our understanding of the composition, degradation and variation of cfDNA in the urinary tract and has laid a foundation for the use of genomewide urinary cfDNA sequencing as a molecular diagnostics tool.

BACKGROUND Recent studies have suggested that single-stranded DNA (ssDNA) library preparation can enrich short DNA species from the plasma of healthy individuals, cancer patients, and transplant recipients. Based on previous observations that fetal DNA molecules in the maternal plasma are shorter than maternal DNA molecules, ssDNA library preparation may potentially enrich fetal DNA and provide substantial improvement in noninvasive prenatal testing. METHODS We tested this hypothesis by comparing the maternal plasma DNA sequencing results using 2 types of ssDNA library preparation methods and a standard double-stranded DNA (dsDNA) library method using samples from first- and third-trimester pregnancies. We also evaluated the performance of ssDNA and dsDNA library methods in the noninvasive prenatal detection of trisomy 21 from maternal plasma. RESULTS Short DNA species were significantly enriched in ssDNA libraries. However, contrary to previous speculation, no significant enrichment was observed in the overall fetal fraction in maternal plasma collected in the first trimester. Our use of an ssDNA library did not reduce the variation in chromosomal representation when compared with a standard dsDNA library in the first-trimester plasma samples. ssDNA libraries also showed inferior performance in the noninvasive prenatal detection of trisomy 21 from maternal plasma. Detailed fetal fraction analysis using size-fractionated Y chromosome sequences and fetal-specific single-nucleotide polymorphisms (SNPs) revealed an unexpected finding that short maternal DNA was preferentially enriched over short fetal DNA in an ssDNA library irrespective of GC content. CONCLUSIONS Our findings have shown that ssDNA library preparation preferentially enriches short maternally derived DNA in maternal plasma.

BACKGROUND Cellular mitochondrial DNA (mtDNA) is organized as circular, covalently closed and double-stranded DNA. Studies have demonstrated the presence of short mtDNA fragments in plasma. It is not known whether circular mtDNA might concurrently exist with linear mtDNA in plasma. METHODS We elucidated the topology of plasma mtDNA using restriction enzyme BfaI cleavage signatures on mtDNA fragment ends to differentiate linear and circular mtDNA. mtDNA fragments with both ends carrying BfaI cleavage signatures were defined as circular-derived mtDNA, whereas those with no cleavage signature or with 1 cleavage signature were defined as linear-derived mtDNA. An independent assay using exonuclease V to remove linear DNA followed by restriction enzyme MspI digestion was used for confirming the conclusions based on BfaI cleavage analysis. We analyzed the presence of BfaI cleavage signatures on plasma DNA ends in nonhematopoietically and hematopoietically derived DNA molecules by sequencing plasma DNA of patients with liver transplantation and bone marrow transplantation. RESULTS Both linear and circular mtDNA coexisted in plasma. In patients with liver transplantation, donor-derived (i.e., liver) mtDNA molecules were mainly linear (median fraction, 91%; range, 75%–97%), whereas recipient-derived (i.e., hematopoietic) mtDNA molecules were mainly circular (median fraction, 88%; range, 77%–93%). The proportion of linear mtDNA was well correlated with liver DNA contribution in the plasma DNA pool (r = 0.83; P value = 0.0008). Consistent data were obtained from a bone marrow transplantation recipient in whom the donor-derived (i.e., hematopoietic) mtDNA molecules were predominantly circular. CONCLUSIONS Linear and circular mtDNA molecules coexist in plasma and may have different tissue origins.

Significance Circulating DNA in plasma has many diagnostic applications, including noninvasive prenatal testing and cancer liquid biopsy. Plasma DNA consists of short fragments of DNA. However, there is little information about mechanisms that are involved in the fragmentation of plasma DNA. We showed that mice in which Dnase1l3 had been deleted showed aberrations in the fragmentation of plasma DNA. We also observed a change in the ranked frequencies of end motifs of plasma DNA caused by the Dnase1l3 deletion. In Dnase1l3−/− mice pregnant with Dnase1l3+/− fetuses, we observed a partial reversal of the plasma DNA aberrations. This study has thus linked the fields of nuclease biology and circulating nucleic acids and has opened up avenues for future research., Circulating DNA in plasma consists of short DNA fragments. The biological processes generating such fragments are not well understood. DNASE1L3 is a secreted DNASE1-like nuclease capable of digesting DNA in chromatin, and its absence causes anti-DNA responses and autoimmunity in humans and mice. We found that the deletion of Dnase1l3 in mice resulted in aberrations in the fragmentation of plasma DNA. Such aberrations included an increase in short DNA molecules below 120 bp, which was positively correlated with anti-DNA antibody levels. We also observed an increase in long, multinucleosomal DNA molecules and decreased frequencies of the most common end motifs found in plasma DNA. These aberrations were independent of anti-DNA response, suggesting that they represented a primary effect of DNASE1L3 loss. Pregnant Dnase1l3−/− mice carrying Dnase1l3+/− fetuses showed a partial restoration of normal frequencies of plasma DNA end motifs, suggesting that DNASE1L3 from Dnase1l3-proficient fetuses could enter maternal systemic circulation and affect both fetal and maternal DNA fragmentation in a systemic as well as local manner. However, the observed shortening of circulating fetal DNA relative to maternal DNA was not affected by the deletion of Dnase1l3. Collectively, our findings demonstrate that DNASE1L3 plays a role in circulating plasma DNA homeostasis by enhancing fragmentation and influencing end-motif frequencies. These results support a distinct role of DNASE1L3 as a regulator of the physical form and availability of cell-free DNA and may have important implications for the mechanism whereby this enzyme prevents autoimmunity.

BACKGROUND Researchers have developed approaches for the noninvasive prenatal testing of single gene diseases. One approach that allows for the noninvasive assessment of both maternally and paternally inherited mutations involves the analysis of single nucleotide polymorphisms (SNPs) in maternal plasma DNA with reference to parental haplotype information. In the past, parental haplotypes were resolved by complex experimental methods or inferential approaches, such as through the analysis of DNA from other affected family members. Recently, microfluidics-based linked-read sequencing technology has become available and allows the direct haplotype phasing of the whole genome rapidly. We explored the feasibility of applying this direct haplotyping technology in noninvasive prenatal testing. METHODS We first resolved the haplotypes of parental genomes with the use of linked-read sequencing technology. Then, we identified SNPs within and flanking the genes of interest in maternal plasma DNA by targeted sequencing. Finally, we applied relative haplotype dosage analysis to deduce the mutation inheritance status of the fetus. RESULTS Haplotype phasing and relative haplotype dosage analysis of 12 out of 13 families were successfully achieved. The mutational status of these 12 fetuses was correctly classified. CONCLUSIONS High-throughput linked-read sequencing followed by maternal plasma-based relative haplotype dosage analysis represents a streamlined approach for noninvasive prenatal testing of inherited single gene diseases. The approach bypasses the need for mutation-specific assays and is not dependent on the availability of DNA from other affected family members. Thus, the approach is universally applicable to pregnancies at risk for the inheritance of a single gene disease.

Background: The presence of fetal DNA in maternal plasma represents a source of fetal genetic material for noninvasive prenatal diagnosis; however, the coexisting background maternal DNA complicates the analysis of aneuploidy in such fetal DNA. Recently, the SERPINB5 gene on chromosome 18 was shown to exhibit different DNA-methylation patterns in the placenta and maternal blood cells, and the allelic ratio for placenta-derived hypomethylated SERPINB5 in maternal plasma was further shown to be useful for noninvasive detection of fetal trisomy 18. Methods: To develop a similar method for the noninvasive detection of trisomy 21, we used methylation-sensitive single nucleotide primer extension and/or bisulfite sequencing to systematically search 114 CpG islands (CGIs)—76% of the 149 CGIs on chromosome 21 identified by bioinformatic criteria—for differentially methylated DNA patterns. The methylation index (MI) of a CpG site was estimated as the proportion of molecules methylated at that site. Results: We identified 22 CGIs which were shown to contain CpG sites that were either completely unmethylated (MI = 0.00) in maternal blood cells and methylated in the placenta (MI range, 0.22–0.65), or completely methylated (MI = 1.00) in maternal blood cells and hypomethylated in the placenta (MI range, 0.00–0.75). We detected, for the first time, placental DNA-methylation patterns on chromosome 21 in maternal plasma during pregnancy and observed their postpartum clearance. Conclusion: Twenty-two (19%) of the 114 studied CGIs on chromosome 21 showed epigenetic differences between samples of placenta and maternal blood cells; these CGIs may provide a rich source of markers for noninvasive prenatal diagnosis.

Severe acute respiratory syndrome (SARS) is the first pandemic of the 21st century (1). Since its recognition, 8437 individuals have been affected and 813 have died (2). Approximately 20–30% of patients required intensive care admission (1). Although there was a slight predominance of female SARS patients, possibly because of the overrepresentation of female healthcare workers (1), male SARS patients were more likely to suffer poor outcomes (3). In a major hospital outbreak in Hong Kong (4), 32% of male and 15% of female SARS patients required intensive care or died. Remarkably, similar demographic data were seen among SARS patients in the greater Toronto area, Canada, where 32% of males and 14% of females with SARS required intensive care or died (5). Karlberg et al. (3) studied the case fatality rates among all confirmed SARS patients documented in the Hong Kong SARS epidemic in 2003. The authors concluded that the mortality rates differed significantly between males and females, being 21.9% and 13.2%, respectively. The relative risk for death in males was 1.62 after adjustment for age. It is thus an intriguing coincidence that ACE2 , the gene for the newly identified functional receptor for the SARS coronavirus, angiotensin-converting enzyme 2, maps to the X-chromosome (Xp22) (6). ACE2 was first identified as a homolog of angiotensin-converting enzyme with zinc metalloproteinase activity (7). Many of its activities differ from those of angiotensin-converting enzyme (8). ACE2 has been found …

BACKGROUND: Spontaneous preterm birth (SPB, before 37 gestational weeks) is a major cause of perinatal mortality and morbidity, but its pathogenesis remains unclear. Studies on SPB have been hampered by the limited availability of markers for SPB in predelivery clinical samples that can be easily compared with gestational age-matched normal controls. We hypothesize that SPB involves aberrant placental RNA expression, and that such RNA transcripts can be detected in predelivery maternal plasma samples, which can be compared with gestational age-matched controls. PRINCIPAL FINDINGS: Using gene expression microarray to profile essentially all human genes, we observed that 426 probe signals were changed by >2.9-fold in the SPB placentas, compared with the spontaneous term birth (STB) placentas. Among the genes represented by those probes, we observed an over-representation of functions in RNA stabilization, extracellular matrix binding, and acute inflammatory response. Using RT-quantitative PCR, we observed differences in the RNA concentrations of certain genes only between the SPB and STB placentas, but not between the STB and term elective cesarean delivery placentas. Notably, 36 RNA transcripts were observed at placental microarray signals higher than a threshold, which indicated the possibility of their detection in maternal plasma. Among them, the IL1RL1 mRNA was tested in plasma samples taken from 37 women. It was detected in 6 of 10 (60%) plasma samples collected during the presentation of preterm labor (≤32.9 weeks) in women eventually giving SPB, but was detected in only 1 of 27 (4%) samples collected during matched gestational weeks from women with no preterm labor (Fisher exact test, p = 0.00056). CONCLUSION: We have identified 36 SPB-associated RNA transcripts, which are possibly detectable in maternal plasma. We have illustrated that the IL1RL1 mRNA was more frequently detected in predelivery maternal plasma samples collected from women resulting in SPB than the gestational-age matched controls.

PURPOSE To compare the early corneal wound repair and inflammatory responses after refractive lenticule extraction (ReLEx) and LASIK. METHODS Eighteen rabbits underwent ReLEx and another 18 underwent LASIK. Each group was divided into three subgroups of six rabbits each and these were subjected to refractive corrections of -3.00 diopters (D), -6.00 D, and -9.00 D. Slit lamp photography, anterior segment optical coherence tomography (AS-OCT), corneal topography, and in vivo confocal microscopy were performed 1 day after surgery. After euthanatization, the corneas were subjected to immunofluorescent staining for fibronectin, CD11b, Ki-67, and TUNEL assay. RESULTS On slit lamp microscopy, all corneas appeared clear pre- and postoperatively in both ReLEx and LASIK eyes. Corneal topography showed a more significant corneal flattening after LASIK than after ReLEx as the degree of correction was increased (P = 0.916 after -3.00 D correction to P = 0.097 after -9.00 D correction). In vivo confocal microscopy showed less light-scattering particles at the flap interface after ReLEx compared with LASIK. Immunostaining of fibronectin showed a less abundant expression in corneas that underwent ReLEx than LASIK. The differences became more marked as the power of correction was increased. Similar trend was seen in the number of CD11b-positive cells (P = 0.476 after -3.00 D correction to P < 0.001 after -9.00D correction). There was no marked disparity observed in cell death and proliferation between post-ReLEx and -LASIK eyes. CONCLUSIONS This study has shown that the ReLEx procedure may result in less topographic changes, inflammation, and early extracellular matrix deposition than LASIK, especially at high refractive correction.

Background: Noninvasive prenatal diagnosis of fetal aneuploidy by maternal plasma analysis is challenging owing to the low fractional and absolute concentrations of fetal DNA in maternal plasma. Previously, we demonstrated for the first time that fetal DNA in maternal plasma could be specifically targeted by epigenetic (DNA methylation) signatures in the placenta. By comparing one such methylated fetal epigenetic marker located on chromosome 21 with another fetal genetic marker located on a reference chromosome in maternal plasma, we could infer the relative dosage of fetal chromosome 21 and noninvasively detect fetal trisomy 21. Here we apply this epigenetic-genetic (EGG) chromosome dosage approach to detect Edwards syndrome (trisomy 18) in the fetus noninvasively. Principal Findings: We have systematically identified methylated fetal epigenetic markers on chromosome 18 by methylated DNA immunoprecipitation (MeDIP) and tiling array analysis with confirmation using quantitative DNA methylation assays. Methylated DNA sequences from an intergenic region between the VAPA and APCDD1 genes (the VAPAAPCDD1 DNA) were detected in pre-delivery, but not post-delivery, maternal plasma samples. The concentrations correlated positively with those of an established fetal genetic marker, ZFY, in pre-delivery maternal plasma. The ratios of methylated VAPA-APCDD1(chr18) to ZFY(chrY) were higher in maternal plasma samples of 9 male trisomy 18 fetuses than those of 27 male euploid fetuses (Mann-Whitney test, P = 0.029). We defined the cutoff value for detecting trisomy 18 fetuses as mean+1.96 SD of the EGG ratios of the euploid cases. Eight of 9 trisomy 18 and 1 of 27 euploid cases showed EGG ratios higher than the cutoff value, giving a sensitivity of 88.9% and a specificity of 96.3%. Conclusions: Our data have shown that the methylated VAPA-APCDD1 DNA in maternal plasma is redominantly derived from the fetus. We have demonstrated that this novel fetal epigenetic marker in maternal plasma is useful for the noninvasive detection of fetal trisomy 18. © Tsui et al., published_or_final_version

The pseudomalignant nature of the placenta prompted us to search for tumor suppressor gene hypermethylation, a phenomenon widely reported in cancer, in the human placenta. Nine tumor suppressor genes were studied. Hypermethylation of the Ras association domain family 1 A (RASSF1A) gene was found in human placentas from all three trimesters of pregnancy but was absent in other fetal tissues. Hypermethylation of Rassf1 was similarly observed in placentas from the rhesus monkey but not the mouse. An inverse relationship between RASSF1A promoter methylation and gene expression was demonstrated by bisulfite sequencing of microdissected placental cells and immunohistochemical staining of placental tissue sections using an anti-RASSF1A antibody. Treatment of choriocarcinoma cell lines, JAR and JEG3, by 5-aza-2′-deoxycytidine and trichostatin A led to reduction in RASSF1A methylation but increased expression. These observations extend the analogy between the primate placenta and malignant tumors to the epigenetic level.

Background: Hirschsprung disease (HSCR) is a congenital disorder characterized by an absence of ganglion cells in the nerve plexuses of the lower digestive tract. HSCR has a complex pattern of inheritance and is sometimes associated with mutations in genes of the receptor tyrosine kinase (RET) and endothelin receptor B (EDNRB) signaling pathways, which are crucial for development of the enteric nervous system. Methods: Using PCR amplification and direct sequencing, we screened for mutations and polymorphisms in the coding regions and intron/exon boundaries of the RET, GDNF, EDNRB, and EDN3 genes of 84 HSCR patients and 96 ethnically matched controls. Results: We identified 10 novel and 2 previously described mutations in RET, and 4 and 2 novel mutations in EDNRB and in EDN3, respectively. Potential disease-causing mutations were detected in 24% of the patients. The overall mutation rate was 41% in females and 19% in males (P = 0.06). RET mutations occurred in 19% of the patients. R114H in RET was the most prevalent mutation, representing 7% of the patients or 37% of the patients with RET mutations. To date, such a high frequency of a single mutation has never been reported in unrelated HSCR patients. Mutations in EDNRB, EDN3, and GDNF were found in four, two, and none of the patients, respectively. Two patients with mutations in genes of the EDNRB pathway also harbored a mutation in RET. Three novel and three reported polymorphisms were found in EDNRB, EDN3, and GDNF. Conclusion: This study identifies additional HSCR disease-causing mutations, some peculiar to the Chinese population, and represents the first comprehensive genetic analysis of sporadic HSCR disease in Chinese.

Dexamethasone (DEX) is a glucocorticoid commonly used in topical eyedrops to treat eye inflammation. It has an undesirable effect of inducing glaucoma in certain patients. In human Trabecular Meshwork (TM) cells DEX regulates a number of genes but its global influence on TM gene expression is still elusive. In the present work, DEX effects on global gene expressions of an established human TM cell line were studied by microarray.The whole experiment of microarray was repeated three times. Differentially expressed genes were identified by an empirical Bayes approach and confirmed by Reverse Transcription Polymerase Chain Reaction.Eight genes (GAS1, CDH4, MT1L, CST3, ATF4, ASNS/TS11, CHOP, HSPA5) were identified that are at least a thousand times more likely to be differentially expressed due to DEX treatment and six genes (TSC22, LDHA, IGFBP2, TAGLN, SCG2, WARS) were identified that are at least a hundred times more likely to be differentially expressed due to DEX treatment. Except for MT1L, ASNS/TS11, IGFBP2, SCG2, and WARS, all the other genes are first reported here to be regulated by DEX in TM. Intriguingly, several of them have overlapping roles in anti-inflammatory response and outflow resistance.The results of our experiments on cultured human TM cells indicate that the increase in outflow resistance and ultimate ocular hypertension may be byproducts of the favorable anti-inflammatory response triggered by DEX.

Purpose The optineurin gene (OPTN) is the second gene besides MYOC in which mutations have been identified to be associated with primary open-angle glaucoma (POAG). In this study, sequence alterations in the OPTN gene associated with POAG in Chinese subjects were investigated. Methods All the coding exons of OPTN were screened, including the intron-exon boundaries, for sequence alterations in a Chinese sample of 119 sporadic patients with POAG and 126 unrelated control subjects by polymerase chain reaction-conformation-sensitive gel electrophoresis and DNA sequencing. Results Sixteen sequence changes were identified: 3 had been reported (T34T, M98K, and R545Q) and 13 were novel (T49T, E103D, V148V, P199P, T202T, H486R, IVS6-5T-->C, IVS6-10G-->A, IVS7+24G-->A, IVS8+20G-->A, IVS13+21C-->G, IVS15+10G-->A, and IVS15-48C-->A). Among them, only E103D, H486R, V148V, and IVS13+21C-->G were found exclusively in patients with POAG, whereas P199P, T202T, and IVS8+20G-->A were present only in control subjects. The genotype of IVS7+24G-->A showed a significant association with POAG (P = 0.02, Fisher two-tailed exact test) and with and increased cup-to-disc ratio in these patients (P = 0.005, Mann-Whitney test). Conclusions The findings in the current study enrich the evidence on the OPTN gene as a causative gene for POAG and suggest a different mutation pattern of OPTN in Chinese than in whites. The wide spectrum of putative mutations detected in this study suggests that both structural and functional disruptions in OPTN may contribute to the pathogenesis of glaucoma.

To discover sequence alterations in the TIGR/MYOC gene associated with primary open-angle glaucoma (POAG) in Chinese subjects.Two hundred one unrelated Chinese patients with POAG and 291 unrelated individuals without glaucoma, aged 50 years or more, were screened for sequence alterations in the TIGR/MYOC gene by polymerase chain reaction, conformation sensitive gel electrophoresis, and DNA sequencing. Up to 111 more control subjects were screened for some of the alterations.Fourteen sequence variants that lead to amino acid changes were identified. Seven were novel: Pro16Leu, Ala17Ser, Leu95Pro, Leu215Pro, Glu300Lys, Glu414Lys, and Tyr471Cys. Of these, Glu300Lys and Tyr471Cys were found only in POAG. Arg46Stop was found in 4 patients with POAG (2.0%) and 9 of 402 control subjects (2.2%); one control subject was homozygous. IOP showed a trend (P = 0.11) toward a decrease of 1.5 mm Hg among the control subjects, with Arg46Stop compared with matched control subjects without Arg46Stop. Gly12Arg occurred four times as frequently in control subjects as in patients, but the difference was not statistically significant.Gly12Arg might be negatively associated with POAG, suggesting a protective effect. Three patients with POAG had a sequence change not found in control subjects, for a frequency of possible disease-causing TIGR/MYOC mutations of 1.5% (95% confidence interval [CI] = 0.3%-4.3%). Arg46Stop occurred with similar frequency in patients with POAG and control subjects, suggesting that the reduced amount of TIGR/MYOC predicted to result from this truncation does not dramatically increase or decrease risk of glaucoma.

Recently published international guidelines recommend the clinical use of noninvasive prenatal test (NIPT) for aneuploidy screening only among pregnant women whose fetuses are deemed at high risk. The applicability of NIPT to aneuploidy screening among average risk pregnancies requires additional supportive evidence. A key determinant of the reliability of aneuploidy NIPT is the fetal DNA fraction in maternal plasma. In this report, we investigated if differences in fetal DNA fractions existed between different pregnancy risk groups. One hundred and ninety-five singleton pregnancies with male fetuses divided into 3 groups according to first trimester screening parameters were examined for fetal DNA percentage by counting Y chromosome DNA sequences using massively parallel sequencing. Fetal DNA fractions were compared between risk groups and assessed for correlations with first trimester screening parameters. There was no statistically significant difference in fetal DNA fractions across the high, intermediate and low risk groups. Fetal DNA fraction showed a strong negative correlation with maternal weight. Fetal DNA fraction also showed weak but significant correlations with gestational age, crown-rump length, multiple of medians of free β-subunit of human chorionic gonadotropin and pregnancy-associated plasma protein A. Similar fetal DNA fractions in maternal plasma between high, intermediate and low risk pregnant women is a precondition for uniform performance of the aneuploidy NIPTs for the general population. This study thus shows that the aneuploidy screening by NIPT is likely to offer similar analytical reliability without respect to the a priori fetal aneuploidy risk.

LASIK (laser-assisted in situ keratomileusis) is a common laser refractive procedure for myopia and astigmatism, involving permanent removal of anterior corneal stromal tissue by excimer ablation beneath a hinged flap. Correction of refractive error is achieved by the resulting change in the curvature of the cornea and is limited by central corneal thickness, as a thin residual stromal bed may result in biomechanical instability of the cornea. A recently developed alternative to LASIK called Refractive Lenticule Extraction (ReLEx) utilizes solely a femtosecond laser (FSL) to incise an intrastromal refractive lenticule (RL), which results in reshaping the corneal curvature and correcting the myopia and/or astigmatism. As the RL is extracted intact in the ReLEx, we hypothesized that it could be cryopreserved and re-implanted at a later date to restore corneal stromal volume, in the event of keratectasia, making ReLEx a potentially reversible procedure, unlike LASIK. In this study, we re-implanted cryopreserved RLs in a non-human primate model of ReLEx. Mild intrastromal haze, noted during the first 2 weeks after re-implantation, subsided after 8 weeks. Refractive parameters including corneal thickness, anterior curvature and refractive error indices were restored to near pre-operative values after the re-implantation. Immunohistochemistry revealed no myofibroblast formation or abnormal collagen type I expression after 8 weeks, and a significant attenuation of fibronectin and tenascin expression from week 8 to 16 after re-implantation. In addition, keratocyte re-population could be found along the implanted RL interfaces. Our findings suggest that RL cryopreservation and re-implantation after ReLEx appears feasible, suggesting the possibility of potential reversibility of the procedure, and possible future uses of RLs in treating other corneal disorders and refractive errors.

PURPOSE. To investigate the coding exons of transforming growth factor (TGF)-β-induced factor (TGIF) for mutations in Chinese patients with high myopia. METHODS. Seventy-one individuals with high myopia of -6.00 1) or less and 105 control subjects were screened by DNA sequencing for sequence alterations. Univariate analysis and logistic regression were performed to identify single-nucleotide polymorphisms (SNPs) and their interactions in TGIF that may he associated with myopia. RESULTS. Six SNPs showed a significant difference (P < 0.05) between patient and control subject in univariate analysis. Four of them cause codon changes: G223R, G231S, P241T, and A262G. Among all the SNPs that entered multivariate analysis, only 657(T→G) showed statistical significance in the logistic regression model (odds ratio 0.133; 95% confidence interval 0.037-0.488; P P = 0.002). CONCLUSIONS. TGIF is a probable candidate gene for high myopia. Further studies are needed to identify, the underlying mechanism.