Application of single-cell RNA-sequencing in ophthalmology / 眼科新进展

Single-cell RNA-sequencing (sc RNA-seq) is a new technique allowing the analysis of transcriptomes in individual cell for discovering specific cell type markers,identifying rare cell types and cellular subsets and revealing the different expression among cells.It is becoming an effective method for researching cellular heterogeneity of complex biological system.In recent years,this approach has been widely used in diverse fields of biology,including stem cell,neurobiology,tumor,immunology,microbiology.In addition,it is also applied in ocular research area.As modem molecular biology and bioinformatics develops,there are some commercial platforms emerging gradually with characteristics of high-throughput,low cost and high efficiency.In this review,we will describe some of the current experimental methods used for sc RNA-seq and discuss the merits and drawbacks,as well as summarize the application of this technique in ophthalmology.In the future,this technique may expand to more studies of ocular-associated diseases for the guidance of clinical diagnosis and therapy.

Phenotype Types and Genetic Mutation Mechanism of Rhesus D Variant Individuals / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the phenotype types and genetic mutation mechanism of Rhesus D variant individuals.</p><p><b>METHODS</b>Fouty-eight peripheral blood samples of pregnancies and blood donors who had been identified as Rhesus D variant by using routine serologic methods were collected from January 2013 to October 2015 in our center. The multiple ligation-dependent probe amplification(MLPA) was used to determine the RHD after genomic DNA had been extracted from the blood sample, then the data including gene copy number variations, point mutations, deletions and hybrid fusions were analyzed by GeneMarker software. All exons of blood sample RHD were amplified via PCR and analyzed by sequencing when its MLPA results were not in accordance with serologic results. Cloning and haplotype sequencing were performed if novel allele had been found.</p><p><b>RESULTS</b>Rh phenotypes of the 48 samples were typed as following: 20 cases out of 48 were CcDee(41.7%, 20/48),12 cases were ccDEe (25%,12/48), 11 cases were CCDee(22.9%, 11/48), 5 cases were CcDEe (10.4%, 5/48), respectively. The MLPA analysis showed that 38 cases possessed only 1 variant allele(RHD zygosity was Dd), while 10 cases possessed 2 variant alleles(RHD zygosity was DD). In Dd type individuals, point mutations were found in 18 cases and RHD/CE hybrid fusions were found in 20 cases. In DDindividuals, point mutations combined with RHD/CE hybrid fusions were found in 9 cases, deletion combined with RHD/CE hybrid fusions were found in 1 case. Variant alleles analysis basing on MLPA showed that 14 cases were weak D 15 and 22 cases were RhD VI type 3, however, the variant alleles were not identified in 7 cases due to lack of detecting probes and were identified via sequencing analysis. Two novel mutations, 79-81delCTC and 689G>A were also certificated by sequencing in 2 cases.</p><p><b>CONCLUSION</b>CcDee is the major Rh phenotype in RhD variants, weak D 15 and RhD VI type 3 are the main serologic type of RhD variants, point mutation and RHD/CE hybrid fusions are main molecular mechanism for RhD variant phenotype. Besides, 79-81delCTC and 689G>A are two novel alleles.</p>

Immunoserology and RHD Genotype Analysis of DVI Type 3 Genotype Pregnant Women with Anti-D / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To performe the immuneserological and RHD Genotype analyses for DVI type 3 genotype pregnemt women with anti-D.</p><p><b>METHODS</b>RhD blood type of this pregnant women was identified by common serological methods, then the blood group specific antibodies was screened and identified; the polymerase chain reaction-sequence specific primer(PCR-SSP) was used to identify the pregnant women's RHD genotype; RhD blood group for the pregnant women, her spouse and daughter was genogrouped and genetically analyzed by multiplex ligation-dependent probe amplification(MLPA). The heredity of this family was analyzed finally.</p><p><b>RESULTS</b>The titer of IgG anti-D in the pregnant woman serum was 1:8; the PCR-SSP showed that the 3rd to 6th exons of RHD gene were missing in the pregnant woman. the genotype of pregnant woman was identified as DVI type 3; the MLPA analysis showed that this pregnant women owned only one RHD allele with 3rd to 6th exons missed, and her genotype was identified as CDe/cde; her spouse was identified as CDe/CDe homozygous genotype, and her daughter as CDe/CDe.</p><p><b>CONCLUSION</b>Accurate identification of RhD blood type is of great significance for a safe and effective clinical blood transfusion strategy, and for taking appropriate measures to prevent hemolytic disease of newborn (HDN) at women childbearing age.</p>

Recent progress in molecular genetics and therapy for X-linked juvenile retinoschisis / 中华实验眼科杂志

Juvenile retinoschisis (RS or XLRS,MIM#312700)is a rare X-linked inherited disorder,mainly affects bilateral retina,and is characterized by cartwheel-like changes of the macular region of the retina and schisis or splitting within the inner retinal layers,leading to visual deterioration.The electroretinogram is beneficial in the diagnosis of juvenile retinoschisis.The a-wave can be of normal or nearly normal amplitude in this disorder,whereas the amplitude of the b-wave is appreciably reduced,giving a decrease in the proportion of b/a.The responsible gene,XLRSl,maps to Xp22 and was identified by positional cloning.This paper makes a brief review about the latest XLRS research of pathogenesis,animal experiments,clinical therapy,and 25 references are cited.

Serological and genetic study of a pedigree featuring a rare p phenotype / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore genetic background of a pedigree with a rare p phenotype from Guangdong province.</p><p><b>METHODS</b>The rare p phenotype was identified by a conventional serologic method. With genomic DNA of proband and family members extracted, exon 3 of alpha-(1,4)galactosyltransferase (A4GALT) gene was amplified with PCR and analyzed by direct sequencing. The mutation found in the pedigree was screened in a normal population using direct sequencing.</p><p><b>RESULTS</b>The proband and 4 family members with the rare p phenotype have all carried a point mutation c.100G>A (p.Val34Ile) in combination with a deletion-insertional mutation c.418_428del11ins34(p.Gln139Trpfs*72), which renders a compound mutation of A4GALT gene. One family member with P2 phenotype has carried a same heterozygous mutation. Of the 100 healthy donors, 5 have carried a heterozygous point mutation c.100G>A, and none carried the deletion-insertional mutation c.418_428del11ins34.</p><p><b>CONCLUSION</b>The rare p phenotype of the pedigree has resulted from a compound mutation of the A4GALT gene, which is in keeping with a recessive inheritance pattern of the p phenotype.</p>

Distribution of bevacizumab in ocular tissue and its toxic effect after injection of anterior chamber / 中华实验眼科杂志

Background Bevacizumab has been widely used in the treatment of new blood vessel disease in ophthalmology.The investigation of the pharmacokinetics and safety after intracameral injection of bevacizumab can offer the basis for the management of iris neovascularization and neovascular glaucoma.Objective The present study was to observe the distribution of bevacizumab(avastin)in eye tissue and toxic effects following the injection of anterior chamber.Methods Twenty-four New Zealand albino rabbits were divided into two groups randomly.0.05 ml (1.25mg)of Bevacizumab was intracamerally injected into the left eyes in the experimental group,and a balanced salt solution of 0.05 ml was injected in the same way into the left eyes of the control group.The anterior segment of eyes and ocular fundus were examined by slit-lamp microscope and direct ophthalmoscope after injection.Intraocular pressure was measured and corneal endothelial microscopy was performed before and after the injections.Five rabbits of the two groups were sacrificed on the first day,the fourth day,the seventh day,the fourteenth day,and the thirtieth day after injection,and the eyeballs were enucleated for histopathological examination.The ultrastructure of eye tissue was observed under the transmission electron microscope on the fourth day and the thirtieth day,and then immunofluorescence staining were performed to assess the distribution of bevacizumab in the eye tissues.This experiment complied with the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission(Version 1988).Results No abnormality in the cornea,lens,vitreous and retina was observed after the injection of bevacizumab under the slit lamp microscope and direct ophthalmoscope.No significant differences were found in intraocular pressure and corneal endothelial cell density in the bevacizumab group compared with the control group before injection and 2 hours,1 day,7 days,14 days,30 days after injection(P =0.760,P =0.956).No histopathological and ultrastructural changes of the cornea,lens,chamber angle,iris,ciliary body and retina were seen after the injection in the experimental group and control group under the light microscope and transmission electron microscope.Bevacizumab was distributed in the anterior chamber angle,iris,ciliary body,choroid and retina in injected eyes and fellow eyes after intracameral injection with red fluorescence and presented the dynamic changes with the lapse of time.The immunofluorescence response of eye tissue to bevacizumab was weaker in the fellow eyes compared with injected eyes.Bevacizumab was mainly distributed in the vessel wall and lumen.Conclusions Bevacizumab can quickly distribute in the vascular tissue of the anterior chamber angle,iris,ciliary body,choroid and retina in injected eyes after intracameral injection without obvious toxic effects to eye tissue.Bevacizumab administered intracamerally may be a new strategy or a joint strategy for iris neovascularisation.