Abstract To investigate the feasibility of constructing parental haplotypes based on short-read next-generation (NGS) and long-read third-generation sequencing (TGS) for non-invasive prenatal testing (NIPT) of thalassemia.Families at risk of having children with moderate to severe thalassemia were recruited for this study. Genomic DNA from both couples and sibling or grandparents of the fetus was first applied to construct haplotypes in couples using targeted NGS. For families where parental haplotypes could not be built using NGS, directly using TGS. NGS was performed on cell-free DNA in maternal peripheral blood to obtain information on fetal allele depth distribution, fetal fraction, etc. Haplotypes and thalassemia genotypes of fetuses were then deduced using the Viterbi decoding algorithm based on a hidden Markov model. Finally, the NIPT results were verified by invasive prenatal diagnosis (IPD). As a result, pedigree-based NGS successfully deduced the thalassemia genotype of the fetus in 93.3%(28/30). However, 2 families had failed because both sibling or grandparents of the fetus and couples were heterozygous for point mutations in the same thalassemia gene. In these 2 families, TGS directly inferred parental haplotypes and deduced fetuses genotypes. This combined strategy resulted in the prediction of fetal genotypes in all 30 families, and its coincidence rate with IPD results reached 100%. In brief, Pedigree-based NGS is enough to construct haplotype and decuce genotypes in most fetuses. TGS is helpful in certain families in which NGS failed. Therefore, comprehensive application of haplotype analysis based on NGS and TGS data is an effective strategy for NIPT in thalassemia.
Intellectual disability (ID) represents a neurodevelopmental disorder, which is characterized by marked defects in the intellectual function and adaptive behavior, with an onset during the developmental period. ID is mainly caused by genetic factors, and it is extremely genetically heterogeneous. This study aims to identify the genetic cause of ID using trio-WES analysis.We recruited four pediatric patients with unexplained ID from non-consanguineous families, who presented at the Department of Pediatrics, Guizhou Provincial People's Hospital. Whole-exome sequencing (WES) and Sanger sequencing validation were performed in the patients and their unaffected parents. Furthermore, conservative analysis and protein structural and functional prediction were performed on the identified pathogenic variants.We identified five novel de novo mutations from four known ID-causing genes in the four included patients, namely COL4A1 (c.2786T>A, p.V929D and c.2797G>A, p.G933S), TBR1 (c.1639_1640insCCCGCAGTCC, p.Y553Sfs*124), CHD7 (c.7013A>T, p.Q2338L), and TUBA1A (c.1350del, p.E450Dfs*34). These mutations were all predicted to be deleterious and were located at highly conserved domains that might affect the structure and function of these proteins.Our findings contribute to expanding the mutational spectrum of ID-related genes and help to deepen the understanding of the genetic causes and heterogeneity of ID.
Rhynchanthus beesianus W. W. Smith, an edible, medicinal, and ornamental plant, is mainly cultivated in China and Myanmar. The essential oil (EO) from R. beesianus rhizome has been used as an aromatic stomachic in China. The chemical composition and biological activities of EO from R. beesianus rhizome were reported for the first time. Based on gas chromatography with flame ionization or mass selective detection (GC-FID/MS) results, the major constituents of EO were 1,8-cineole (47.6%), borneol (15.0%), methyleugenol (11.2%), and bornyl formate (7.6%). For bioactivities, EO showed a significant antibacterial activity against Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Proteus vulgaris with the diameter of the inhibition zone (DIZ) (8.66–10.56 mm), minimal inhibitory concentration (MIC) (3.13–6.25 mg/mL), and minimal bactericidal concentration (MBC) (6.25–12.5 mg/mL). Moreover, EO (128 μg/mL) significantly inhibited the production of proinflammatory mediators nitric oxide (NO) (92.73 ± 1.50%) and cytokines tumor necrosis factor-α (TNF-α) (20.29 ± 0.17%) and interleukin-6 (IL-6) (61.08 ± 0.13%) in lipopolysaccharide (LPS)-induced RAW264.7 macrophages without any cytotoxic effect. Moreover, EO exhibited significant acetylcholinesterase (AChE) inhibitory activity (the concentration of the sample that affords a 50% inhibition in the assay (IC50) = 1.03 ± 0.18 mg/mL) and moderate α-glucosidase inhibition effect (IC50 = 11.60 ± 0.25 mg/mL). Thus, the EO could be regarded as a bioactive natural product and has a high exploitation potential in the cosmetics and pharmaceutical industries.
A 6-month-old female infant presented with unexplained hemolytic anemia, showing no abnormalities by capillary electrophoresis and genetic testing for α- and β-thalassemia mutations that are commonly seen in the Chinese population. A rare Hb Mizuho: [HBB: c.206T > C β 68(E12) Leu- Pro] variant was identified by next-generation sequencing (NGS) and verified by Sanger sequencing. Hb Mizuho: [HBB: c.206T > C β 68(E12) Leu- Pro] is not easily detectable because it is extremely unstable, and the correct diagnosis is usually made via DNA sequencing. This is the first report of this variant in the Chinese population.
Background: Guizhou Province is an area with high incidence of thalassemia. However, there are few large-sample studies on the correlation between genotypes and phenotypes in Guizhou Province. In this study, the phenotypes and genotypes of 1174 patients with thalassemia in Guizhou Province were collected, and the relationship between different genotypes and phenotypes was analyzed, providing a more accurate basis for genetic counseling, prevention and control of thalassemia. Methods: A total of 1174 patients with thalassemia were collected in Guizhou Provincial People's Hospital from October 2020 to December 2021 by PCR-reverse dot blot (RDB) hybridization assay, and their red blood cell (RBC), hemoglobin (Hb), mean erythrocyte volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), hemoglobin (HbA), hemoglobin A2 (HbA2), and fetal hemoglobin (HbF) data were collected. The relationship between different genotypes and phenotypes was analyzed. Results: Among 1174 cases of thalassemia or carriers, there were 617 cases of a-thalassemia, 512 cases of b-thalassemia, 45 cases of coinheritance of aand b-tha-lassemia. The severity of anemia between a-thalassemia was positively correlated with the decrease of non-functional copy number of a-globin gene. The degree of anemia in non-deletion a-thalassemia was greater than that in deletion a-thalassemia. In b-thalassemia, b0 gene mutation did not produce b-globin, and b+ mutation expressed some bglobin, but it was lower than normal level. b0/b0 had no bglobin production, and long-term blood transfusion was required to maintain life. Compared with a-thalassemia, the degree of anemia in b-thalassemia whose clinical type was same as a-thalassemia was more serious. The anemia degree of coinheritance of aand b-thalassemia was less than that of simple a-thalassemia or b-thalassemia. Conclusions: The clinical phenotype of thalassemia is influenced by molecular mechanism, and the two kinds of thalassemia can interact with each other. The clinical severity is positively correlated with the imbalance of a peptide chain and b peptide chain. A comprehensive understanding of the hematologic phenotype differences between different genotypes and subtypes of thalassemia can provide more accurate data for genetic counseling of thalassemia.
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