Case report: Preimplantation genetic testing for X-linked alport syndrome caused by variation in the COL4A5 gene

X-Linked Alport Syndrome (XLAS) is an X-linked, dominant, hereditary nephropathy mainly caused by mutations in the COL4A5 gene, found on chromosome Xq22. In this study, we reported a pedigree with XLAS caused by a COL4A5 mutation. This family gave birth to a boy with XLAS who developed hematuria and proteinuria at the age of 1 year. We used next-generation sequencing (NGS) to identify mutations in the proband and his parents and confirmed the results using Sanger sequencing. This testing showed there was a single nucleotide missense variation, c.3659G>A (p.Gly1220Asp) (NM_033380.3), in the COL4A5 gene. To prevent the inheritance of the syndrome, we used eight embryos for trophoblast biopsy after assisted reproductive technology treatment, and whole genome amplification (WGA) was performed using multiple annealing and looping-based amplification cycles (MALBAC). Embryos were subjected to Preimplantation Genetic Testing (PGT) procedures, including Sanger sequencing, NGS-based single nucleotide polymorphism (SNP) haplotype linkage analysis, and chromosomal copy number variation (CNV) analysis. The results showed that three embryos (E1, E2, and E4) were free of CNV and genetic variation in the COL4A5 gene. Embryo E1 (4AA) was transferred after consideration of the embryo growth rate, morphology, and PGT results. Prenatal diagnosis in the second trimester showed that the fetus had a normal karyotype and did not carry the COL4A5 mutation (c.3659G>A). Ultimately, a healthy boy was born and did not carry the pathogenic COL4A5 mutation, which indicated that PGT prevented the intergenerational transmission of the causative mutation of XLAS

Establishment of linkage phase, using Oxford Nanopore Technologies, for preimplantation genetic testing of Coffin-Lowry syndrome with a de novo RPS6KA3 mutation

Background: This study aimed to perform preimplantation genetic testing (PGT) for a female Coffin-Lowry Syndrome (CLS) patient with a de novo mutation (DNM) in RPS6KA3. It was challenging to establish the haplotype in this family because of the lack of information from affected family members. Hence, we explored a new and reliable strategy for the detection of the DNM in PGT, using Oxford Nanopore Technologies (ONT) and the MARSALA platform.Methods: We performed whole-exome sequencing (WES) on the proband and confirmed the pathogenic mutation by Sanger sequencing. The proband then underwent PGT to prevent the transmission of the pathogenic mutation to her offspring. We diverged from the conventional methods and used long-read sequencing (LRS) on the ONT platform to directly detect the mutation and nearby SNPs, for construction of the haplotype in the preclinical phase of PGT. In the clinical phase of embryo diagnosis, the MARSALA method was used to detect both the SNP-based haplotype and chromosome copy number variations (CNVs), in each blastocyst. Finally, a normal embryo was selected by comparison to the haplotype of the proband and transferred into the uterus. Sanger sequencing and karyotyping were performed by amniocentesis, at 17 weeks of gestation, to confirm the accuracy of PGT.Results: Using WES, we found the novel, heterozygous, pathogenic c.1496delG (p.Gly499Valfs*25) mutation of RPS6KA3 in the proband. The SNP-based haplotype that was linked to the pathogenic mutation site was successfully established in the proband, without the need for other family members to be tested with ONT. Eight blastocysts were biopsied to perform PGT and were assessed with a haplotype linkage analysis (30 SNP sites selected), to give results that were consistent with direct mutation detection using Sanger sequencing. The results of PGT showed that three of the eight blastocysts were normal, without the DNM. Moreover, the patient had a successful pregnancy, after transfer of a normal blastocyst into the uterus, and delivered a healthy baby.Conclusion: The ONT platform, combined with the MARSALA method, can be used to perform PGT for DNM patients without the need for other samples as a reference

Response of Nitrogen Metabolism in Masson Pine Needles to Elevated CO2

To explore the response of nitrogen metabolism in Masson pine (Pinus massoniana) to high CO2 concentrations, needles from one-year-old seedlings were used as materials to detect key enzyme activities, gene expression and different forms of nitrogen metabolites after CO2 stress for different durations (0 h, 6 h, 12 h, 24 h). The results show that elevated CO2 affected the efficiency of nitrogen metabolism in Masson pine needles, inhibiting the expression of key genes involved in nitrogen metabolism, including glutamate synthase (GOGAT), nitrite reductase (NiR), glutamine synthase (GS), nitrate reductase (NR) and glutamate dehydrogenase (GDH), and decreasing the activities of GOGAT, NiR, and GS. The decrease in enzyme activities and gene expression caused a decrease in different forms of nitrogen metabolites, including total nitrogen, ammonium, nitrite and specific amino acids. With prolonged stress, the nitrate content increased first and then decreased. In this study, the response pattern of nitrogen metabolism to CO2 stress in Masson pine needles was described, which may aid future research on nitrogen utilization in Masson pine

Transcriptional Analysis of Masson Pine (Pinus massoniana) under High CO2 Stress

To explore the molecular mechanism of the response of Masson pine (Pinus massoniana), the main coniferous tree in southern China, to high CO2 stress, transcriptome sequencing was carried out to analyze the genome-wide responses of annual seedlings under different durations (0 h, 6 h, 12 h and 24 h) of high CO2 stress. The results showed that a total of 3080/1908, 3110/2115 and 2684/1483 genes were up-/down-regulated after 6 h, 12 h and 24 h of treatment, respectively, compared with control check group (CK, 0 h). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that most of these differentially expressed genes (DEGs) were enriched in energy metabolism, carbohydrate synthesis, cell wall precursor synthesis and hormone regulation pathways. For energy metabolism, the expression of most genes involved in photosynthesis (including the light reaction and Calvin cycle) was generally inhibited, while the expression of genes related glycolysis, the tricarboxylic acid (TCA) cycle and PPP pathway was up-regulated. In addition, the increase in the CO2 concentration induced the up-regulation of gene expression in the sucrose synthesis pathway. Among all starch synthesis genes, GBSS (granule-bound starch synthase) had the highest expression level. On the other hand, during the synthesis of hemicellulose and pectin (cell wall precursor substances), the expression levels of GMD (GDP-mannose 4,6-dehydratase), MGP (Mannose-1-phosphate guanylyl transferase) and RHM (Rhamnose biosynthetic enzyme) were the highest, suggesting that the synthesis of the raw materials hemicellulose and pectin in Masson pine under stress were mainly supplied by GDP-Man, GDP-Fuc and UDP-Rha. Finally, stress inhibited gene expression in the ABA (Abscisic Acid) synthesis pathway and induced gene expression in the GA (Gibberellin), SA (Salicylic acid), BR(Brassinolide) and MeJA (Methyl Jasmonate) pathways. Stomatal switches were regulated by hormonal interactions. This experiment elaborated on the response and molecular mechanism of Masson pine to CO2 stress and aided in screening carbon sequestration genes for the corresponding molecular research of Masson pine in the future

Effects of Endophytic Fungi on Seedling Growth and Phosphorus Uptake of Mytilaria laosensis Under Phosphorus Stress

【Objective】The study was conducted to explore the effects of endophytic fungi on phosphorus uptake in Mytilaria Laosensis seedlings under low phosphorus stress, and to select the endophytic fungi that could promote the growth and phosphorus uptake in M. Laosensis seedlings.【Method】Taking M. Laosensis seedlings as the research object, 10 strains (M1, M2, M3, M4, M5, M6, M7, M8, M9, M10) were inoculated by the method of irrigating bacterial solution. Fifteen days later, low phosphorus stress test (18, 12, 6, 0 mg/kg) was conducted, after sixty days of low phosphorus stress, the height and ground diameter, the root dry weight and the phosphorus content of the shoots and roots of plants, the acid phosphatase activity in leaves and roots and rhizosphere soil were measured to explore the growth and phosphorus uptake of M. Laosensis.【Result】With the increase of stress degree, strain M9 and M10 could increase the height, ground diameter and root-shoot ratio of M. Laosensis and promote its growth. Under normal conditions and severe stress, strain M6 could effectively increase the phosphorus content in shoot of M. Laosensis by 36. 9% and 41. 1%, respectively. With the increase of stress degree, strain M9 had a more prominent effect and strain M4 could significantly increase the phosphorus content in the root of M. Laosensis, which increased by 57.7% under severe stress. Under severe stress, both strains M6 and M9 could significantly increase the acid phosphatase activity in leaves of M. Laosensis and soil; except for mild stress, the acid phosphatase activity of the root system of M. Laosensis treated with the strain was significantly higher than that of CK.【Conclusion】Under soil phosphorus stress, inoculation of endophytic fungi has a significant effect on the growth and phosphorus uptake of M. Laosensis. Under different stress environments, strains M6 and M9 can increase phosphorus content, acid phosphatase activity and superoxide dismutase activity in M. Laosensis to a certain extent and promote seedling growth efficiently