Abnormal morphology and function in retinal ganglion cells derived from patients-specific iPSCs generated from individuals with Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease that results from degeneration of retinal ganglion cells (RGC). Mitochondrial ND4 11778G > A mutation, which affects structural components of complex I, is the most prevalent LHON-associated mitochondrial DNA (mtDNA) mutation worldwide. The m.11778G > A mutation is the primary contributor underlying the development of LHON and X-linked PRICKLE3 allele (c.157C > T, p.Arg53Trp) linked to biogenesis of ATPase interacts with m.11778G > A mutation to cause LHON. However, the lack of appropriate cell and animal models of LHON has been significant obstacles for deep elucidation of disease pathophysiology, specifically the tissue-specific effects. Using RGC-like cells differentiated from induced pluripotent stem cells (iPSCs) from members of one Chinese family (asymptomatic subjects carrying only m.11778G > A mutation or PRICKLE3 p.Arg53Trp mutation, symptomatic individuals bearing both m.11778G > A and PRICKLE3 p.Arg53Trp mutations and control lacking these mutations), we demonstrated the deleterious effects of mitochondrial dysfunctions on the morphology and functions of RGCs. Notably, iPSCs bearing only m.11778G > A or p.Arg53Trp mutation exhibited mild defects in differentiation to RGC-like cells. The RGC-like cells carrying only m.11778G > A or p.Arg53Trp mutation displayed mild defects in RGC morphology, including the area of soma and numbers of neurites, electrophysiological properties, ATP contents and apoptosis. Strikingly, those RGC-like cells derived from symptomatic individuals harboring both m.11778G > A and p.Arg53Trp mutations displayed greater defects in the development, morphology and functions than those in cells bearing single mutation. These findings provide new insights into pathophysiology of LHON arising from RGC deficiencies caused by synergy between m.11778G > A and PRICKLE3 p.Arg53Trp mutation.

Dysfunction of VIPR2 leads to myopia in humans and mice.

BACKGROUND: Myopia is the leading cause of refractive errors. As its pathogenesis is poorly understood, we determined if the retinal VIP-VIPR2 signalling pathway axis has a role in controlling signalling output that affects myopia development in mice. METHODS: Association analysis meta-study, single-cell transcriptome, bulk RNA sequencing, pharmacological manipulation and VIPR2 gene knockout studies were used to clarify if changes in the VIP-VIPR2 signalling pathway affect refractive development in mice. RESULTS: The SNP rs6979985 of the VIPR2 gene was associated with high myopia in a Chinese Han cohort (randomceffect model: p=0.013). After either 1 or 2 days' form deprivation (FD) retinal VIP mRNA expression was downregulated. Retinal single-cell transcriptome sequencing showed that VIPR2 was expressed mainly by bipolar cells. Furthermore, the cAMP signalling pathway axis was inhibited in some VIPR2+ clusters after 2 days of FD. The selective VIPR2 antagonist PG99-465 induced relative myopia, whereas the selective VIPR2 agonist Ro25-1553 inhibited this response. In Vipr2 knockout (Vipr2-KO) mice, refraction was significantly shifted towards myopia (p<0.05). The amplitudes of the bipolar cell derived b-waves in 7-week-old Vipr2-KO mice were significantly larger than those in their WT littermates (p<0.05). CONCLUSIONS: Loss of VIPR2 function likely compromises bipolar cell function based on presumed changes in signal transduction due to altered signature electrical wave activity output in these mice. As these effects correspond with increases in form deprivation myopia (FDM), the VIP-VIPR2 signalling pathway axis is a viable novel target to control the development of this condition.

Form-deprivation myopia downregulates calcium levels in retinal horizontal cells in mice.

The process of eye axis lengthening in myopic eyes is regulated by multiple mechanisms in the retina, and horizontal cells (HCs) are an essential interneuron in the visual regulatory system. Wherein intracellular Ca2+ plays an important role in the events involved in the regulatory role of HCs in the retinal neural network. It is unknown if intracellular Ca2+ regulation in HCs mediates changes in the retinal neural network during myopia progression. We describe here a novel calcium fluorescence indicator system that monitors HCs' intracellular Ca2+ levels during form-deprivation myopia (FDM) in mice. AAV injection of GCaMP6s, as a protein calcium sensor, into a Gja10-Cre mouse monitored the changes in Ca2+signaling in HC that accompany FDM progression in mice. An alternative Gja10-Cre/Ai96-GCaMP6s mouse model was created by cross mating Gja10-Cre with Ai96 mice. Immunofluorescence imaging and live imaging of the retinal cells verified the identity of these animal models. Changes in retinal horizontal cellular Ca2+ levels were resolved during FDM development. The numbers of GCaMP6s and the proportion of HCs were tracked based on profiling changes in GCaMP6s+calbindin+/calbindin+ coimmunostaining patterns. They significantly decreased more after either two days (P < 0.01) or two weeks (P < 0.001) in form deprived eyes than in the untreated fellow eyes. These decreases in their proportion reached significance only in the retinal central region rather than also in the retinal periphery. A novel approach employing a GCaMP6s mouse model was developed that may ultimately clarify if HCs mediate Ca2+ signals that contribute to controlling FDM progression in mice. The results indicate so far that FDM progression is associated with declines in HC Ca2+ signaling activity.

Genetic Interaction between Mfrp and Adipor1 Mutations Affect Retinal Disease Phenotypes.

Adipor1tm1Dgen and Mfrprd6 mutant mice share similar eye disease characteristics. Previously, studies established a functional relationship of ADIPOR1 and MFRP proteins in maintaining retinal lipidome homeostasis and visual function. However, the independent and/or interactive contribution of both genes to similar disease phenotypes, including fundus spots, decreased axial length, and photoreceptor degeneration has yet to be examined. We performed a gene-interaction study where homozygous Adipor1tm1Dgen and Mfrprd6 mice were bred together and the resulting doubly heterozygous F1 offspring were intercrossed to produce 210 F2 progeny. Four-month-old mice from all nine genotypic combinations obtained in the F2 generation were assessed for white spots by fundus photo documentation, for axial length by caliper measurements, and for photoreceptor degeneration by histology. Two-way factorial ANOVA was performed to study individual as well as gene interaction effects on each phenotype. Here, we report the first observation of reduced axial length in Adipor1tmlDgen homozygotes. We show that while Adipor1 and Mfrp interact to affect spotting and degeneration, they act independently to control axial length, highlighting the complex functional association between these two genes. Further examination of the molecular basis of this interaction may help in uncovering mechanisms by which these genes perturb ocular homeostasis.

Declines in PDE4B activity promote myopia progression through downregulation of scleral collagen expression.

Myopia is the most common cause of a visual refractive error worldwide. Cyclic adenosine monophosphate (cAMP)-linked signaling pathways contribute to the regulation of myopia development, and increases in cAMP accumulation promote myopia progression. To pinpoint the underlying mechanisms by which cAMP modulates myopia progression, we performed scleral transcriptome sequencing analysis in form-deprived mice, a well-established model of myopia development. Form deprivation significantly inhibited the expression levels of genes in the cAMP catabolic pathway. Quantitative real-time polymerase chain reaction analysis validated that the gene expression level of phosphodiesterase 4B (PDE4B), a cAMP hydrolase, was downregulated in form-deprived mouse eyes. Under visually unobstructed conditions, loss of PDE4B function in Pde4b-knockout mice increased the myopic shift in refraction, -3.661 ± 1.071 diopters, more than that in the Pde4b-wildtype littermates (P < 0.05). This suggests that downregulation and inhibition of PDE4B gives rise to myopia. In guinea pigs, subconjunctival injection of rolipram, a selective inhibitor of PDE4, led to myopia in normal eyes, and it also enhanced form-deprivation myopia (FDM). Subconjunctival injection of dibutyryl-cyclic adenosine monophosphate, a cAMP analog, induced only a myopic shift in the normal visually unobstructed eyes, but it did not enhance FDM. As myopia developed, axial elongation occurred during scleral remodeling that was correlated with changes in collagen fibril thickness and distribution. The median collagen fibril diameter in the FDM + rolipram group, 55.09 ± 1.83 nm, was thinner than in the FDM + vehicle group, 59.33 ± 2.06 nm (P = 0.011). Thus, inhibition of PDE4 activity with rolipram thinned the collagen fibril diameter relative to the vehicle treatment in form-deprived eyes. Rolipram also inhibited increases in collagen synthesis induced by TGF-ß2 in cultured human scleral fibroblasts. The current results further support a role for PDE enzymes such as PDE4B in the regulation of normal refractive development and myopia because either loss or inhibition of PDE4B function increased myopia and FDM development through declines in the scleral collagen fibril diameter.

The exome sequencing identified the mutation in YARS2 encoding the mitochondrial tyrosyl-tRNA synthetase as a nuclear modifier for the phenotypic manifestation of Leber's hereditary optic neuropathy-associated mitochondrial DNA mutation.

Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disorder. Nuclear modifier genes are proposed to modify the phenotypic expression of LHON-associated mitochondrial DNA (mtDNA) mutations. By using an exome sequencing approach, we identified a LHON susceptibility allele (c.572G>T, p.191Gly>Val) in YARS2 gene encoding mitochondrial tyrosyl-tRNA synthetase, which interacts with m.11778G>A mutation to cause visual failure. We performed functional assays by using lymphoblastoid cell lines derived from members of Chinese families (asymptomatic individuals carrying m.11778G>A mutation, or both m.11778G>A and heterozygous p.191Gly>Val mutations and symptomatic subjects harboring m.11778G>A and homozygous p.191Gly>Val mutations) and controls lacking these mutations. The 191Gly>Val mutation reduced the YARS2 protein level in the mutant cells. The aminoacylated efficiency and steady-state level of tRNA(Tyr) were markedly decreased in the cell lines derived from patients both carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The failure in tRNA(Tyr) metabolism impaired mitochondrial translation, especially for polypeptides with high content of tyrosine codon such as ND4, ND5, ND6 and COX2 in cells lines carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The YARS2 p.191Gly>Val mutation worsened the respiratory phenotypes associated with m.11778G>A mutation, especially reducing activities of complexes I and IV. The respiratory deficiency altered the efficiency of mitochondrial ATP synthesis and increased the production of reactive oxygen species. Thus, mutated YARS2 aggravates mitochondrial dysfunctions associated with the m.11778G>A mutation, exceeding the threshold for the expression of blindness phenotype. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between mtDNA mutation and mutated nuclear-modifier YARS2.

A genome-wide meta-analysis identifies two novel loci associated with high myopia in the Han Chinese population.

High myopia, highly prevalent in the Chinese population, is a leading cause of visual impairment worldwide. Genetic factors play a critical role in the development of this visual disorder. Genome-wide association studies in recent years have revealed several chromosomal regions that contribute to its progression. To identify additional genetic variants for high myopia susceptibility, we used a genome-wide meta-analysis to examine the associations between the disease and 286 031 single-nucleotide polymorphisms (SNPs) in a combined cohort of 665 cases and 960 controls. The most significant SNPs (n = 61) were genotyped in a replication cohort (850 cases and 1197 controls), and 14 SNPs were further tested through genotyping in two additional validation cohorts (combined 1278 cases and 2486 controls). As a result of this analysis, four SNPs reached genome-wide significance (P < 2.0 × 10(-7)). The most significantly associated SNP, rs2730260 [overall P = 8.95 × 10(-14); odds ratio (95% CI) =1.33 (1.23-1.44)], is located in the VIPR2 gene, which is located in the MYP4 locus. The other three SNPs (rs7839488, rs4395927 and rs4455882) in the same linkage disequilibrium block are located in the SNTB1 gene, with -P values ranging from 1.13 × 10(-8) to 2.13 × 10(-11). The VIPR2 and SNTB1 genes are expressed in the retina and the retinal pigment epithelium and have been previously reported to have potential functions for the pathogenesis of myopia. Our results suggest that variants of the VIPR2 and SNTB1 genes increase susceptibility to high myopia in Han Chinese.

[A novel mutation T8821G in mitochondrial DNA may be associated with Leber's hereditary optic neuropathy].

OBJECTIVE: To report on clinical, genetic and molecular characterization of two Chinese families with Leber's hereditary optic neuropathy. METHODS: Ophthalmological examinations have revealed variable severity and age at onset of visual loss among the probands and other matrilineal relatives of both families. The entire mitochondrial genome of the two probands was amplified with PCR in 24 overlapping fragments using sets of oligonucleotide primers. RESULTS: The ophthalmological examinations showed that penetrance was 12.5% and 30.0% respectively in the two families. Sequence analysis of the complete mitochondrial genomes in these pedigrees has identified unreported homoplasmic T8821G mutation in the ATPase 6 gene and distinct sets of polymorphisms belonging to haplogroups M10a. The T8821G mutation has occurred at the extremely conserved nucleotide (conventional position 99) of the ATPase6. Thus, this mutation may alter structural formation of ATPase6, thereby leading to failure in the synthesis of ATP involved in visual impairment. CONCLUSION: Above observations have suggested that the ATPase6 T8821G mutation may be involved in the pathogenesis of optic neuropathy in these families.

Genetic variants at 13q12.12 are associated with high myopia in the Han Chinese population.

High myopia, which is extremely prevalent in the Chinese population, is one of the leading causes of blindness in the world. Genetic factors play a critical role in the development of the condition. To identify the genetic variants associated with high myopia in the Han Chinese, we conducted a genome-wide association study (GWAS) of 493,947 SNPs in 1088 individuals (419 cases and 669 controls) from a Han Chinese cohort and followed up on signals that were associated with p < 1.0 × 10(-4) in three independent cohorts (combined, 2803 cases and 5642 controls). We identified a significant association between high myopia and a variant at 13q12.12 (rs9318086, combined p = 1.91 × 10(-16), heterozygous odds ratio = 1.32, and homozygous odds ratio = 1.64). Furthermore, five additional SNPs (rs9510902, rs3794338, rs1886970, rs7325450, and rs7331047) in the same linkage disequilibrium (LD) block with rs9318086 also proved to be significantly associated with high myopia in the Han Chinese population; p values ranged from 5.46 × 10(-11) to 6.16 × 10(-16). This associated locus contains three genes-MIPEP, C1QTNF9B-AS1, and C1QTNF9B. MIPEP and C1QTNF9B were found to be expressed in the retina and retinal pigment epithelium (RPE) and are more likely than C1QTNF9B-AS1 to be associated with high myopia given the evidence of retinal signaling that controls eye growth. Our results suggest that the variants at 13q12.12 are associated with high myopia.

Mitochondrial haplotypes may modulate the phenotypic manifestation of the LHON-associated ND1 G3460A mutation in Chinese families.

To investigate the pathophysiology of Leber's hereditary optic neuropathy (LHON), a cohort of 1164 Han Chinese subjects with LHON were screened for ND1 G3460A mutation. A total of 295 subjects from 16 Han Chinese families carrying the G3460A mutation underwent a clinical and genetic evaluation and molecular analysis of mitochondrial (mt)DNA. The incidence of G3460A mutation was 1.4% in this cohort of Chinese subjects with LHON. Twenty-seven (20 males/7 females) of 109 matrilineal relatives among 10 Chinese pedigrees carrying this mutation exhibited a wide range of severity and age-at-onset in visual impairment. Penetrances of optic neuropathy ranged from 7.1% to 50%, with the average of 24.5%. The age-at-onset of 27 affected matrilineal relatives varied from 10 to 40 years, with the average of 22 years. Molecular analysis identified the homoplasmic G3460A mutation and distinct sets of variants belonging to eight haplogroups. Haplogroup M with G3460A mutation was of higher frequency than those in controls. The penetrances of visual loss in families carrying mitochondrial DNA haplogroups A, B and M were higher than those in other families. Furthermore, haplogroup-specific variants tRNA(Ser(AGY)) A12223G, tRNA(Thr) G15927A and tRNA(Glu) A14693G may enhance the penetrance of visual loss in these families. The G3460A mutation occurred through recurrent origins and founder events in Chinese population. Mitochondrial modifiers may modulate the penetrance and expressivity of optic neuropathy among Chinese pedigrees carrying the G3460A mutation. Thus, our findings may provide new insights into the understanding of pathophysiology and valuable information on the management of LHON.

[The analysis of mitochondrial DNA haplogroups and variants for Leber's hereditary optic neuropathy in Chinese families carrying the m.14484T >C mutation].

The m.14484T>C mutation in mitochondrial ND6 gene (MT-ND6) is a primary mutation underlying the development of Leber's hereditary optic neuropathy (LHON) , but by itself not enough to cause visual loss. To explore the role of mitochondrial haplogroups on the expression of LHON for the people carrying the m.14484T>C mutation, we performed systematic and extended mutational screening of MT-ND6 gene in a cohort of 1177 Han Chinese patients with LHON. A total of 67 affected subjects carried the homoplasmic m.14484T>C mutation, accounting for 5.7% of this LHON population. The penetrances of optic neuropathy among 51 pedigrees carrying the m.14484T>C mutation ranged from 5.6% to 100.0%, with the average of 21.5%. The sequence analysis of entire mitochondrial genomes of 51 probands exhibited distinct sets of polymorphisms belonging to 18 Eastern Asian haplogroups. The frequencies of haplogroup A and haplogroup F were sig-nificantly less in the LHON mtDNA samples than those in 106 Chinese controls. On the other hand, the haplogroup M10a accounted for 9.8% of the patient's mtDNA samples but was absent in 106 Chinese controls. Strikingly, the average pene-trance (46.13%) of optic neuropathy for the pedigrees carrying mitochondrial haplogroup M10a was higher than those car-rying other mtDNA haplogroups. These observations indicated that mitochondrial haplogroup M10a may increase the risk of visual loss.

Conformal Fabrication of Thick Film Platinum Strain Gauge Via Error Regulation Strategies for In Situ High-Temperature Strain Detection.

Monitoring high-temperature strain on curved components in harsh environments is a challenge for a wide range of applications, including in aircraft engines, gas turbines, and hypersonic vehicles. Although there are significant improvements in the preparation of high-temperature piezoresistive film on planar surfaces using 3D printing methods, there are still difficulties with poor surface compatibility and high-temperature strain testing on curved surfaces. Herein, a conformal direct ink writing (CDIW) system coupled with an error feedback regulation strategy was used to fabricate high-precision, thick films on curved surfaces. This strategy enabled the maximum amount of error in the distance between the needle and the substrate on a curved surface to be regulated from 155 to 4 µm. A conformal Pt thick-film strain gauge (CPTFSG) with a room-temperature strain coefficient of 1.7 was created on a curved metallic substrate for the first time. The resistance drift rate at 800 °C for 1 h was 1.1%, which demonstrated the excellent stability and oxidation resistance of the CPTFSG. High-temperature dynamic strain tests up to 769 °C revealed that the sensor had excellent high-temperature strain test performance. Furthermore, the CPTFSG was conformally deposited on an aero-engine turbine blade to perform in situ tensile and compressive strain testing at room temperature. High-temperature strain tests were conducted at 100 and 200 °C for 600 and 580 µÎµ, respectively, demonstrating a high steady-state response consistent with the commercial high-temperature strain transducer. In addition, steady-state strain tests at high temperatures up to 496 °C were tested. The CDIW error modulation strategy provides a highly promising approach for the high-precision fabrication of Pt thick films on complex surfaces and driving in situ sensing of high-temperature parameters on curved components toward practical applications.

Rapid laser fabrication of indium tin oxide and polymer-derived ceramic composite thin films for high-temperature sensors.

Thin-film sensors are essential for real-time monitoring of components in high-temperature environments. Traditional fabrication methods often involve complicated fabrication steps or require prolonged high-temperature annealing, limiting their practical applicability. Here, we present an approach using direct ink writing and laser scanning (DIW-LS) to fabricate high-temperature functional thin films. An indium tin oxide (ITO)/preceramic polymer (PP) ink suitable for DIW was developed. Under LS, the ITO/PP thin film shrank in volume. Meanwhile, the rapid pyrolysis of PP into amorphous precursor-derived ceramic (PDC) facilitated the faster sintering of ITO nanoparticles and improved the densification of the thin film. This process realized the formation of a conductive network of interconnected ITO nanoparticles. The results show that the ITO/PDC thin film exhibits excellent stability, with a drift rate of 4.7 % at 1000 °C for 25 h, and withstands temperatures up to 1250 °C in the ambient atmosphere. It is also sensitive to strain, with a maximum gauge factor of -6.0. As a proof of concept, we have used DIW-LS technology to fabricate a thin-film heat flux sensor on the surface of the turbine blade, capable of measuring heat flux densities over 1 MW/m2. This DIW-LS process provides a viable approach for the integrated, rapid, and flexible fabrication of thin film sensors for harsh environments.

Exome sequencing reveals CCDC111 mutation associated with high myopia.

Myopia is a refractive error of the eye that is prevalent worldwide. The most extreme form, high myopia, is usually associated with other ocular disorders such as retinal detachment, macular degeneration, cataract, and glaucoma, and is one of leading causes of blindness. The etiology is complex and has not been fully elucidated. In this study, we identified a novel missense variant of the CCDC111 gene (NM_152683.2: c.265T > G; p.Y89D) in a high myopia family by exome sequencing. The variant was identified in 4 patients from an additional 270 sporadic high myopia patients, but not found in 270 controls. The amino acid is highly conserved across species, and variants giving rise to amino acid substitutions are predicted to be functionally damaging. The CCDC111 gene was ubiquitously expressed in primary cell cultures from human eye tissue, including corneal epithelial cells, choroidal melanoma cells, scleral fibroblasts, retinal epithelial cells, retinal Müller cells, and lens capsule epithelial cells. In summary, our results suggested that the CCDC111 may be a susceptibility gene for high myopia.

Thin-Film Platinum Resistance Temperature Detector with a SiCN/Yttria-Stabilized Zirconia Protective Layer by Direct Ink Writing for High-Temperature Applications.

In situ temperature monitoring of curved high-temperature components in extreme environments is challenging for a variety of applications in fields such as aero engines and gas turbines. Recently, extrusion-based direct ink writing (DIW) has been utilized to fabricate platinum (Pt) resistance temperature detectors (RTDs). However, the current Pt RTD prepared by DIW technology suffers from a limited temperature range and poor high-temperature stability. Here, DIW technology and yttria-stabilized zirconia (YSZ)-modified precursor ceramic film packaging have been used to build a Pt RTD with high-temperature resistance, small disturbance, and high stability. The results indicate that the protective layer formed by the liquid phase anchors the Pt particles and reduces the agglomeration and volatilization of the Pt sensitive layer at high temperature. Attributed to the SiCN/YSZ protective layer, the temperature resistance curve of the Pt RTD in the range of 50-800 °C has little deviation from the fitting curve, and the fitting correlation coefficient is above 0.9999. Interestingly, the Pt RTD also has high repeatability and stability. The high temperature resistance drift rate is only 0.05%/h after 100 h of long-term testing at 800 °C and can withstand butane flame up to ∼1300 °C without damage. Moreover, the Pt RTD can be conformally deposited on the outer ring of aerospace bearings by DIW technology and then realize on-site, nondestructive, and real-time monitoring of bearing temperature. The fabricated Pt RTD shows great potential for high-temperature applications, and the novel technology proposed provides a feasible pathway for temperature monitoring of aeroengine internal curved hot-end components.

All-Three-Dimensionally-Printed AgPd Thick-Film Strain Gauge with a Glass-Ceramic Protective Layer for High-Temperature Applications.

A high-temperature thin/thick-film strain gauge (TFSG) shows development prospects for in situ strain monitoring of hot-end components due to their small perturbations, no damage, and fast response. Direct ink writing (DIW) 3D printing is an emerging and facile approach for the rapid fabrication of TFSG. However, TFSGs prepared based on 3D printing with both high thermal stability and low temperature coefficient of resistance (TCR) over a wide temperature range remain a great challenge. Here, we report a AgPd TFSG with a glass-ceramic protective layer based on DIW. By encapsulating the AgPd sensitive layer and regulating the Pd content, the AgPd TFSG demonstrated a low TCR (191.6 ppm/°C) from 50 to 800 °C and ultrahigh stability (with a resistance drift rate of 0.14%/h at 800 °C). Meanwhile, the achieved specifications for strain detection included a strain sensing range of ±500 µÎµ, fast response time of 153 ms, gauge factor of 0.75 at 800 °C, and high durability of >8000 cyclic loading tests. The AgPd TFSG effectively monitors strain in superalloys and can be directly deposited onto cylindrical surfaces, demonstrating the scalability of the presented approach. This work provides a strategy to develop TFSGs for in situ sensing of complex curved surfaces in harsh environments.

Experimental murine myopia induces collagen type Iα1 (COL1A1) DNA methylation and altered COL1A1 messenger RNA expression in sclera.

PURPOSE: To investigate whether myopia development is associated with changes of scleral DNA methylation in cytosine-phosphate-guanine (CpG) sites in the collagen 1A1 (COL1A1) promoter and messenger RNA (mRNA) levels following murine form deprivation myopia. METHODS: Fifty-seven C57BL/6 mice (postnatal day 23) were randomly assigned to four groups: (1) monocular form deprivation (MD) in which a diffuser lens was placed over one eye for 28 days; (2) normal controls without MD; (3) MD recovery in which the diffuser lens was removed for seven days; and (4) MD recovery normal controls. The DNA methylation pattern in COL1A1 promoter and exon 1 was determined by bisulfite DNA sequencing, and the COL1A1 mRNA level in sclera was determined by quantitative PCR. RESULTS: MD was found to induce myopia in the treated eyes. Six CpG sites in the promoter and exon 1 region of COL1A1 were methylated with significantly higher frequency in the treated eyes than normal control eyes (p<0.05), with CpG island methylation in MD-contralateral eyes being intermediate. Consistent with the CpG methylation, scleral COL1A1 mRNA was reduced by 57% in the MD-treated eyes compared to normal controls (p<0.05). After seven days of MD recovery, CpG methylation was significantly reduced (p=0.01). The methylation patterns returned to near normal level in five CpG sites, but the sixth was hypomethylated compared to normal controls. CONCLUSIONS: In parallel with the development of myopia and the reduced COL1A1 mRNA, the frequency of methylation in CpG sites of the COL1A1 promoter/exon 1 increased during MD and returned to near normal during recovery. Thus, hypermethylation of CpG sites in the promoter/exon 1 of COL1A1 may underlie reduced collagen synthesis at the transcriptional level in myopic scleras.

[The analysis of Leber's hereditary optic neuropathy associated with mitochondrial tRNAAla C5601T mutation in seven Han Chinese families].

We reported here the clinical, genetic, and molecular characterization of Leber's hereditary optic neuropathy (LHON) with C5601T mutation in seven Chinese families. The ophthalmologic examinations of seven Chinese families who were clinically diagnosed LHON were conducted. Strikingly, these families exhibited very low penetrance of visual impairment, and the penetrance was 9.5%, 14.3%, 4.5%, 8.3%, 10.0%, 22.2% and 25.0%. Meanwhile, entire mitochondrial genome of seven probands was amplified by PCR using 24 pairs of oligonucleotide primers with overlapping fragments. Molecular analysis of mitochondrial DNA (mtDNA) in these pedigrees revealed the absence of three common LHON associated G11778A, G3460A and T14484C mutations but the presence of homoplastic LHON associated tRNAAla C5601T mutation in probands and other matrilineal relatives. These mtDNA polymorphism sites belongs to the Asian haplogroups G2, G2a1, G2a1, G2, G2b, G2a1 and G2. By analyzing mitochondrial genome, seven LHON families all carry the C5601T mutation. The C5601T mutation occurs at the highly conserved nucleotide (conventional position 59) of tRNAAla, thereby contributing to the structural formation and stabilization of functional tRNAs and leading to mitochondrial dysfunction involved in visual impairment. The incomplete penetrance of visual loss in these seven Chinese pedigrees strongly indicates that the tRNAAla C5601T mutation was itself insufficient to produce a clinical phenotype. The lack of functional mtDNA variants in these pedigrees ruled out the role of mitochondrial background in the phenotypic expression of visual loss. Therefore, nuclear backgrounds and environmental factors seem to be modifying factors for the phenotypic manifestation of the tRNAAla C5601T mutation in the seven Chinese families.

Common Postzygotic Mutational Signatures in Healthy Adult Tissues Related to Embryonic Hypoxia.

Postzygotic mutations are acquired in normal tissues throughout an individual's lifetime and hold clues for identifying mutagenic factors. Here, we investigated postzygotic mutation spectra of healthy individuals using optimized ultra-deep exome sequencing of the time-series samples from the same volunteer as well as the samples from different individuals. In blood, sperm, and muscle cells, we resolved three common types of mutational signatures. Signatures A and B represent clock-like mutational processes, and the polymorphisms of epigenetic regulation genes influence the proportion of signature B in mutation profiles. Notably, signature C, characterized by C>T transitions at GpCpN sites, tends to be a feature of diverse normal tissues. Mutations of this type are likely to occur early during embryonic development, supported by their relatively high allelic frequencies, presence in multiple tissues, and decrease in occurrence with age. Almost none of the public datasets for tumors feature this signature, except for 19.6% of samples of clear cell renal cell carcinoma with increased activation of the hypoxia-inducible factor 1 (HIF-1) signaling pathway. Moreover, the accumulation of signature C in the mutation profile was accelerated in a human embryonic stem cell line with drug-induced activation of HIF-1α. Thus, embryonic hypoxia may explain this novel signature across multiple normal tissues. Our study suggests that hypoxic condition in an early stage of embryonic development is a crucial factor inducing C>T transitions at GpCpN sites; and individuals' genetic background may also influence their postzygotic mutation profiles.

Low penetrance of Leber's hereditary optic neuropathy in ten Han Chinese families carrying the ND6 T11484C mutation.

BACKGROUND: Leber's hereditary optic neuropathy (LHON) is a maternally inherited disorder. The purpose of this investigation is to understand the role of mitochondrial haplotypes in the development of LHON associated with ND6 T14484C mutation in Chinese families. METHODS: One hundred fourteen subjects from ten Han Chinese families with LHON were studied by the clinical and genetic evaluation as well as molecular and biochemical analyses of mitochondrial DNA (mtDNA). RESULTS: Clinical evaluation revealed that ten families exhibited extremely low penetrance of visual impairment, with an average of 10%. In particular, ten (8 males/2 females) of 114 matrilineal relatives in these families exhibited the variable severity and age-at-onset in visual dysfunction. The average age-of-onset of vision loss was 19 years old. Molecular analysis of mitochondrial DNA (mtDNA) identified the homoplasmic T14484C mutation and distinct sets of variants, belonging to the Asian haplogroups B5b, D4, D4g1b, G3a2, R11, R11a and Z3, respectively. However, there was the absence of secondary LHON-associated mtDNA mutations in these ten Chinese families. CONCLUSION: The low penetrance of vision loss in these Chinese pedigrees strongly indicated that the T14484C mutation was itself insufficient to produce a clinical phenotype. The absence of secondary LHON mtDNA mutations suggests that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the T14484C mutation in those Chinese families with low penentrace of vision loss. However, nuclear modifier genes and environmental factors appear to be modifier factors for the phenotypic manifestation of the T14484C mutation in these Chinese families.