A Family Screening of CD19 Gene Mutation by Restriction Fragment Length Polymorphism

Background: CD19 molecule found on B lymphocyte surface forms CD19 complex together with CD21, CD81, CD225 in mature B cells and regulates B lymphocyte activation with antigen stimulation. Mutation(s) in the gene encoding the CD19 molecule affect CD19 protein expression and primary immunodeficiency (PID) occurs. Some genetic method, especially sanger and next generation sequencing and flow cytometric methods are widely used in the diagnosis of PID. The RFLP method, which is faster and cheaper than other mutation detection methods, is rarely used in the diagnosis of PID. The study aimed to genetically identify CD19 deficiency, which is a PID, using the RFLP method. Methods: The study was performed at Necmettin Erbakan University, Meram Medicine Faculty Hospital, Pediatric Allergy and Immunology clinic. A total of 8 patients and two healthy controls could be included in the study. A total of 8 patients and two healthy controls were included in the study, and the relevant region genotypes in the CD19 gene were determined by performing RCR-RFLP analysis. Results: CD19 deficiency was first described by us. The index case, newborn baby and mother were also included in the study. It was determined that the index case (P6) was homozygous mutant, the newborn baby (P7) and mother (P8) had heterozygous genotype. Based on this situation, one child (P1) was found to be homozygous mutant, mother (P2), father (P3) and other children (P4 and P5) had heterozygous genotype in the family, which was determined to be related to the first case. Conclusion: Rapid genetic diagnosis in patients suspected of having a known case of PID insufficiency as a result of clinical and laboratory findings carries a vital risk in terms of treatment options to be offered to patients. Although PCR-RFLP, which is a cheap, safe and fast method, is used to detect known mutations, the use of PID is rare. In our study, it has been shown that it is a method that can be used in the diagnosis of PID by determining genotypes using PCR-RFLP, and especially in terms of rapid genetic testing of family screenings.

Generation of a New Glutinous Photothermosensitive Genic-Male-Sterile (PTGMS) Line by CRISPR/Cas9-Directed Mutagenesis of Wx in Rice (Oryza sativa L.)

The Photothermosensitive Genic-Male-Sterile (PTGMS) line, Y58S, an indica rice variety, combines high-quality and high-light-efficiency use, disease and stress resistance, and excellent plant type and mating force. Y58S is widely used to assemble two-line hybrid rice varieties, especially super hybrids. The Wx gene is the main effector gene for controlling amylose synthesis, which determines the amylose content (AC) of rice grains. By editing this gene, a glutinous line with a low AC can be obtained. In this study, the CRISPR/Cas9 system was used to mediate the editing of the Wx gene, which caused ultra-low AC mutations that produced a PTGMS glutinous rice strain with excellent waxiness. The results showed that 18 positively transformed plants were obtained from the T0 generation, with a mutation rate of 64.29%, of which six were homozygous mutant plants, indicating that the gene-editing target had a higher targeting efficiency and a higher homozygosity mutation rate. Compared to the wild type, the AC of the mutants was significantly lower. Through molecular marker detection and screening of T1 and T2 generations, five homozygous T-DNA-free mutant strains were identified that were consistent with Y58S in fertility and other agronomic traits except for AC. Among these, the AC of the W-1-B-5 homozygous mutant, the glutinous PTGMS line wx-Y58S, was as low as 0.6%. Our research revealed that the Wx gene of excellent PTGMS rice can be edited to generate a new waxy PTGMS line using the CRISPR/Cas9 system. This study provided a simple and effective strategy for breeding high-yield, high-quality, and glutinous two-line hybrid rice, and provided excellent sterile lines for their large-scale application. Once put into use, waxy hybrid rice will greatly improve the yield of glutinous rice and increase social benefits.

Detection of Urinary Exosomal HSD11B2 mRNA Expression: A Useful Novel Tool for the Diagnostic Approach of Dysfunctional 11β-HSD2-Related Hypertension

ObjectiveApparent mineralocorticoid excess (AME) is an autosomal recessive disorder caused by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) enzyme deficiency, traditionally assessed by measuring either the urinary cortisol metabolites ratio (tetrahydrocortisol+allotetrahydrocortisol/tetrahydrocortisone, THF+5αTHF/THE) or the urinary cortisol/cortisone (F/E) ratio. Exosomal mRNA is an emerging diagnostic tool due to its stability in body fluids and its biological regulatory function. It is unknown whether urinary exosomal HSD11B2 mRNA is related to steroid ratio or the HSD11B2 662 C>G genotype (corresponding to a 221 A>G substitution) in patients with AME and essential hypertension (EH).Aim of the StudyTo detect and quantify HSD11B2 mRNA from urinary exosomes in samples from family members affected by AME and EH, and to evaluate the relationship between exosomal HSD11B2 mRNA, steroid ratio, 662C>G genotype, and hypertension.MethodsIn this observational case–control study, urinary steroid ratios and biochemical parameters were measured. Urinary exosomes were extracted from urine and exosomal HSD11B2 mRNA was quantified by Droplet Digital PCR (ddPCR). B2M (β-2 microglobulin) gene was selected as the reference housekeeping gene.ResultsAmong family members affected by AME, exosomal urinary HSD11B2 mRNA expression was strictly related to genotypes. The two homozygous mutant probands showed the highest HSD11B2 mRNA levels (median 169, range 118–220 copies/µl) that progressively decreased in 221 AG heterozygous with hypertension (108, range 92–124 copies/µl), 221 AG heterozygous normotensives (23.35, range 8–38.7 copies/µl), and wild-type 221 AA subjects (5.5, range 4.5–14 copies/µl). Heterozygous hypertensive subjects had more HSD11B2 mRNA than heterozygous normotensive subjects. The F/E urinary ratio correlated with HSD11B2 mRNA copy number (p < 0.05); HSD11B2 mRNA strongly decreased while THF+5αTHF/THE increased in the two probands after therapy. In the AME family, HSD11B2 copy number correlated with both F/E and THF+5αTHF/THE ratios, whereas in EH patients, a high F/E ratio reflected a reduced HSD11B2 mRNA expression.ConclusionsHSD11B2 mRNA is detectable and quantifiable in urinary exosomes; its expression varies according to the 662 C>G genotype with the highest levels in homozygous mutant subjects. The HSD11B2 mRNA overexpression in AME could be due to a compensatory mechanism of the enzyme impairment. Exosomal mRNA is a useful tool to investigate HSD11B2 dysregulation in hypertension.

Deletion of the SELENOP gene leads to CNS atrophy with cerebellar ataxia in dogs

We investigated a hereditary cerebellar ataxia in Belgian Shepherd dogs. Affected dogs developed uncoordinated movements and intention tremor at two weeks of age. The severity of clinical signs was highly variable. Histopathology demonstrated atrophy of the CNS, particularly in the cerebellum. Combined linkage and homozygosity mapping in a family with four affected puppies delineated a 52 Mb critical interval. The comparison of whole genome sequence data of one affected dog to 735 control genomes revealed a private homozygous structural variant in the critical interval, chr4:66,946,539_66,963,863del17,325. This deletion includes the entire protein coding sequence of SELENOP and is predicted to result in complete absence of the encoded selenoprotein P required for selenium transport into the CNS. Genotypes at the deletion showed the expected co-segregation with the phenotype in the investigated family. Total selenium levels in the blood of homozygous mutant puppies of the investigated litter were reduced to about 30% of the value of a homozygous wildtype littermate. Genotyping >600 Belgian Shepherd dogs revealed an additional homozygous mutant dog. This dog also suffered from pronounced ataxia, but reached an age of 10 years. Selenop-/- knock-out mice were reported to develop ataxia, but their histopathological changes were less severe than in the investigated dogs. Our results demonstrate that deletion of the SELENOP gene in dogs cause a defect in selenium transport associated with CNS atrophy and cerebellar ataxia (CACA). The affected dogs represent a valuable spontaneous animal model to gain further insights into the pathophysiological consequences of CNS selenium deficiency.

Retinal neovascularization induced by mutant Vldlr gene inhibited in an inherited retinitis pigmentosa mouse model: an in-vivo study

AIM: To explore whether the retinal neovascularization (NV) in a genetic mutant mice model could be ameliorated in an inherited retinitis pigmentosa (RP) mouse, which would help to elucidate the possible mechanism and prevention of retinal NV diseases in clinic. METHODS: The Vldlr-/- mice, the genetic mutant mouse model of retinal NV caused by the homozygous mutation of Vldlr gene, with the rd1 mice, the inherited RP mouse caused by homozygous mutation of Pde6b gene were bred. Intercrossing of the above two mice led to the birth of the F1 hybrids, further inbreeding of which gave birth to the F2 offspring. The ocular genotypes and phenotypes of the mice from all generations were examined, with the F2 offspring grouped according to the genotypes. RESULTS: The rd1 mice exhibited the RP phenotype of outer retinal degeneration and loss of retinal function. The Vldlr-/- mice exhibited the phenotype of retinal NV obviously shown by the fundus fluorescein angiography. The F1 hydrides, with the heterozygote genotype, exhibited no phenotypes of RP or retinal NV. The F2 offspring with homozygous genotypes were grouped into four subgroups. They were the F2-I mice with the wild-type Pde6b and Vldlr genes (Pde6b+/+-Vldlr+/+), which had normal ocular phenotypes; the F2-II mice with homozygous mutant Vldlr gene (Pde6b+/+-Vldlr-/-), which exhibited the retinal NV phenotype; the F2-III mice with homozygous mutant Pde6b gene (Pde6b-/--Vldlr+/+), which exhibited the RP phenotype. Specifically, the F2-IV mice with homozygous mutant Vldlr and Pde6b gene (Pde6b-/--Vldlr-/-) showed only the RP phenotype, without the signs of retinal NV. CONCLUSION: The retinal NV can be inhibited by the RP phenotype, which implies the role of a hyperoxic state in treating retinal NV diseases.

Effects of Myostatin Mutation on Onset of Laying, Egg Production, Fertility, and Hatchability

Increased body weight and muscle mass, along with improved feed efficiency, by myostatin (MSTN) mutation in quail, supports the potential use of MSTN as a selection marker for higher meat yield in the poultry industry. Although economically important traits of broilers have been studied using recently generated MSTN mutant quail, the effect of MSTN mutation on egg production has not yet been investigated. In this study, several economically important traits of layers, including egg production, reproduction, and body composition of hens, were compared between MSTN homozygous mutant, heterozygous mutant, and wild-type (WT) quail. In terms of egg production, MSTN homozygous mutant quail, showing significantly delayed onset of egg laying, laid significantly heavier eggs, but a significantly lower number of eggs compared to WT quail for 20 days after 3 months of age, resulting in similar total egg production among groups. In addition, the percentage proportion of egg white and yolk in egg weight were similar among groups. Furthermore, similar fertility and hatchability of eggs from MSTN homozygous mutant breeding pairs and WT breeding pairs indicated normal reproductive function of MSTN mutant quail. These findings will provide scientific rationales for the consideration of MSTN as a potential selection marker for layers in the poultry industry.

Altered germline cyst formation and oogenesis in Tex14 mutant mice

ABSTRACT During oocyte differentiation in mouse fetal ovaries, sister germ cells are connected by intercellular bridges, forming germline cysts. Within the cyst, primary oocytes form via gaining cytoplasm and organelles from sister germ cells through germ cell connectivity. To uncover the role of intercellular bridges in oocyte differentiation, we analyzed mutant female mice lacking testis-expressed 14 (TEX14), a protein involved in intercellular bridge formation and stabilization. In Tex14 homozygous mutant fetal ovaries, germ cells divide to form a reduced number of cysts in which germ cells remained connected via syncytia or fragmented cell membranes, rather than normal intercellular bridges. Compared with wild-type cysts, homozygous mutant cysts fragmented at a higher frequency and produced a greatly reduced number of primary oocytes with precocious cytoplasmic enrichment and enlarged volume. By contrast, Tex14 heterozygous mutant germline cysts were less fragmented and generate primary oocytes at a reduced size. Moreover, enlarged primary oocytes in homozygous mutants were used more efficiently to sustain folliculogenesis than undersized heterozygous mutant primary oocytes. Our observations directly link the nature of fetal germline cysts to oocyte differentiation and development.

A midbody component homolog, too much information/prc1-like, is required for microtubule reorganization during both cytokinesis and axis induction in the early zebrafish embryo

We show that the zebrafish maternal-effect mutation too much information (tmi) corresponds to zebrafish prc1-like (prc1l), which encodes a member of the MAP65/Ase1/PRC1family of microtubule-associated proteins. Embryos from tmi/prc1l homozygous mutant mothers display cytokinesis defects in meiotic and mitotic divisions in the early embryo, indicating that tmi/prc1l has a role in midbody formation during cell division at the egg-to-embryo transition. Unexpectedly, maternal tmi/prc1l function is also essential for the reorganization of vegetal pole microtubules required for embryonic axis induction. While Prc1 is widely regarded to crosslink microtubules in an antiparallel conformation, our studies provide evidence for an additional function of Prc1 in the bundling of parallel microtubules in the vegetal cortex of the early embryo during cortical rotation and prior to mitotic cycling. These findings highlight common yet distinct aspects of microtubule reorganization that occur during the egg-to-embryo transition, driven by maternal product for the midbody component Prc1l and required for embryonic cell division and pattern formation.

Production of High Amylose and Resistant Starch Rice Through Targeted Mutagenesis of Starch Branching Enzyme Iib by Crispr/cas9

Rice is the staple food for half of the world’s population. Starch accounts for 80-90% of the total mass of rice seeds, and rice starch is low in resistant starch (RS) with a high glycemic index (GI). RS has gained important since it is beneficial in preventing various diseases. Starch branching enzyme IIb (SBEIIb) plays a key role in the amylopectin synthesis pathway in the endosperm of cereals. Down-regulation of SBEIIb in several important crops has led to high amylose, high RS and low GI starch. In this study, we mutated OsSBEIIb in the japonica rice cultivar TNG82 through CRISPR/Cas9 and investigated the molecular and physicochemical modifications in OsSBEIIb mutant lines, e.g., gene expression, enzyme activity, apparent amylose content (AAC), RS and GI. As expected, the levels of modification in these starch related traits in heterozygous mutant lines were about half as those of homozygous mutant lines. Gene expression and enzyme activity of OsSBEIIb were down-regulated significantly while AAC and RS contents increased progressively from 17.4% and 0.5% in WT, respectively, to as high as 25.0% and 7.5% in heterozygous mutant lines and 36.0% and 12.0% in homozygous mutant lines. Consequently, with increased RS and decreased rate of reducing sugar production, GI progressively decreased in heterozygous and homozygous mutant rice endosperms by 11% and 28%, respectively. Our results demonstrate that it has huge potential for precise and efficient generation of high RS and low GI rice through CRISPR/Cas9 to provide a more suitable source of starch for type II diabetes.

CRISPR/Cas9 Targeted Mutagenesis for Functional Genetics in Maize

The CRISPR/Cas9-based system for targeted mutagenesis has become an indispensable tool for functional genetics in plants. CRISPR/Cas9 allows users to generate loss-of-function alleles in genes of interest with precision and in a simple-to-use system. This manuscript outlines important points to consider for experimental design and utilization of CRISPR/Cas9 in targeted mutagenesis in maize. It also introduces the pRGEB32-BAR vector modified for use in maize that allows simultaneous delivery of multiple gRNAs using a simple assembly. Vector selection, gRNA design, genetic strategies, and genotyping approaches are discussed, with an emphasis on achieving isolation of homozygous mutant plants in a time- and cost-efficient manner.