The frequency of HKαα allele in silent deletional α-thalassemia carriers in the Yulin region of southern China using the third-generation sequencing.

OBJECTIVES: α-thalassemia is relatively prevalent in Yulin Region in southern China. In order to accurately detect α-globin gene aberrations for genetic counseling, the prevalence of HKαα (Hong Kong αα) allele in this subpopulation of silent deletional α-thalassemia were examined. MATERIALS AND METHODS: A total of 1845 subjects were selected in Yulin Region from January 2021 to March 2021. Peripheral blood was collected from each participant for routine genetic analysis of thalassemia. The HKαα allele was determined using the Single-molecule real-time (SMRT) technology for samples with -α3.7/αα, ßN/ßN genotype. RESULTS: Two samples were identified with HKαα allele from 100 samples with -α3.7/αα, ßN/ßN genotype. The frequency of HKαα allele was 2.0 % (2/100) in -α3.7/αα, ßN/ßN carriers in Yulin Region. One sample was identified with a novel variant of the α-globin gene cluster named αHKαα by SMRT technology. One rare HBA2 variant and six HBB variants were found by SMRT technology, including -α3.7/HBA2:c.300 + 34G > A, HBB:c.316-45G > C/ßN, HBB:c.315 + 180 T > C/ßN, HBB:c.316-179A > C/ßN. CONCLUSION: A certain proportion of HKαα allele had been detected in Yulin Region. SMRT technology plays a crucial role for improving the diagnostic accuracy and positive detection rate of thalassemia. The completion of this study has great meaning for strengthening the prevention and control of thalassemia in Yulin Region.

High-throughput site-specific N-glycoproteomics reveals glyco-signatures for liver disease diagnosis.

The glycoproteome has emerged as a prominent target for screening biomarkers, as altered glycosylation is a hallmark of cancer cells. In this work, we incorporated tandem mass tag labeling into quantitative glycoproteomics by developing a chemical labeling-assisted complementary dissociation method for the multiplexed analysis of intact N-glycopeptides. Benefiting from the complementary nature of two different mass spectrometry dissociation methods for identification and multiplex labeling for quantification of intact N-glycopeptides, we conducted the most comprehensive site-specific and subclass-specific N-glycosylation profiling of human serum immunoglobulin G (IgG) to date. By analysing the serum of 90 human patients with varying severities of liver diseases, as well as healthy controls, we identified that the combination of IgG1-H3N5F1 and IgG4-H4N3 can be used for distinguishing between different stages of liver diseases. Finally, we used targeted parallel reaction monitoring to successfully validate the expression changes of glycosylation in liver diseases in a different sample cohort that included 45 serum samples.

Cancer-associated fibroblast related gene signature in Helicobacter pylori-based subtypes of gastric carcinoma for prognosis and tumor microenvironment estimation in silico analysis.

Introduction: Gastric cancer (GC) remains the major constituent of cancer-related deaths and a global public health challenge with a high incidence rate. Helicobacter pylori (HP) plays an essential role in promoting the occurrence and progression of GC. Cancer-associated fibroblasts (CAFs) are regarded as a significant component in the tumor microenvironment (TME), which is related to the metastasis of GC. However, the regulation mechanisms of CAFs in HP-related GC are not elucidated thoroughly. Methods: HP-related genes (HRGs) were downloaded from the GSE84437 and TCGA-GC databases. The two databases were combined into one cohort for training. Furthermore, the consensus unsupervised clustering analysis was obtained to sort the training cohort into different groups for the identification of differential expression genes (DEGs). Weighted correlation network analysis (WGCNA) was performed to verify the correlation between the DEGs and cancer-associated fibroblasts which were key components in the tumor microenvironment. The least absolute shrinkage and selection operator (LASSO) was executed to find cancer-associated fibroblast-related differential expression genes (CDEGs) for the further establishment of a prognostic model. Results and discussion: In this study, 52 HP-related genes (HRGs) were screened out based on the GSE84437 and TCGA-GC databases. A total of 804 GC samples were analyzed, respectively, and clustered into two HP-related subtypes. The DEGs identified from the two subtypes were proved to have a relationship with TME. After WGCNA and LASSO, the CAFs-related module was identified, from which 21 gene signatures were confirmed. Then, a CDEGs-Score was constructed and its prediction efficiency in GC patients was conducted for validation. Overall, a highly precise nomogram was established for enhancing the adaptability of the CDEGs-Score. Furthermore, our findings revealed the applicability of CDEGs-Score in the sensitivity of chemotherapeutic drugs. In general, our research provided brand-new possibilities for comprehending HP-related GC, evaluating survival, and more efficient therapeutic strategies.

Back-to-Back Comparison of Third-Generation Sequencing and Next-Generation Sequencing in Carrier Screening of Thalassemia.

CONTEXT.­: Recently, new technologies, such as next-generation sequencing and third-generation sequencing, have been used in carrier screening of thalassemia. However, there is no direct comparison between the 2 methods in carrier screening of thalassemia. OBJECTIVE.­: To compare the clinical performance of third-generation sequencing with next-generation sequencing in carrier screening of thalassemia. DESIGN.­: Next-generation sequencing and third-generation sequencing were simultaneously conducted for 1122 individuals in Hainan Province. RESULTS.­: Among 1122 genetic results, 1105 (98.48%) were concordant and 17 (1.52%) were discordant between the 2 methods. Among the 17 discordant results, 4 were common thalassemia variants, 9 were rare thalassemia variants, and 4 were variations with unknown pathogenicity. Sanger sequencing and polymerase chain reaction for discordant samples confirmed all the results of third-generation sequencing. Among the 685 individuals with common and rare thalassemia variants detected by third-generation sequencing, 512 (74.74%) were carriers of α-thalassemia, 110 (16.06%) were carriers of ß-thalassemia, and 63 (9.20%) had coinheritance of α-thalassemia and ß-thalassemia. Three thalassemia variants were reported for the first time in Hainan Province, including -THAI, -α2.4, and ααααanti3.7. Eleven variants with potential pathogenicity were identified in 36 patients with positive hemoglobin test results. Among 52 individuals with negative hemoglobin test results, 17 were identified with thalassemia variants. In total, third-generation sequencing and next-generation sequencing correctly detected 763 and 746 individuals with variants, respectively. Third-generation sequencing yielded a 2.28% (17 of 746) increment compared with next-generation sequencing. CONCLUSIONS.­: Third-generation sequencing was demonstrated to be a more accurate and reliable approach in carrier screening of thalassemia compared with next-generation sequencing.

Third-Generation Sequencing as a New Comprehensive Technology for Identifying Rare α- and ß-Globin Gene Variants in Thalassemia Alleles in the Chinese Population.

CONTEXT.­: Identification of rare thalassemia variants requires a combination of multiple diagnostic technologies. OBJECTIVE.­: To investigate a new approach of comprehensive analysis of thalassemia alleles based on third-generation sequencing (TGS) for identification of α- and ß-globin gene variants. DESIGN.­: Enrolled in this study were 70 suspected carriers of rare thalassemia variants. Routine gap-polymerase chain reaction and DNA sequencing were used to detect rare thalassemia variants, and TGS technology was performed to identify α- and ß-globin gene variants. RESULTS.­: Twenty-three cases that carried rare variants in α- and ß-globin genes were identified by the routine detection methods. TGS technology yielded a 7.14% (5 of 70) increment of rare α- and ß-globin gene variants as compared with the routine methods. Among them, the rare deletional genotype of -THAI was the most common variant. In addition, rare variants of CD15 (G>A) (HBA2:c.46G>A), CD117/118(+TCA) (HBA1:c.354_355insTCA), and ß-thalassemia 3.5-kilobase gene deletion were first identified in Fujian Province, China; to the best of our knowledge, this is the second report in the Chinese population. Moreover, HBA1:c.-24C>G, IVS-II-55 (G>T) (HBA1:c.300+55G>T) and hemoglobin (Hb) Maranon (HBA2:c.94A>G) were first identified in the Chinese population. We also identified rare Hb variants of HbC, HbG-Honolulu, Hb Miyashiro, and HbG-Coushatta in this study. CONCLUSIONS.­: TGS technology can effectively and accurately detect deletional and nondeletional thalassemia variants simultaneously in one experiment. Our study also demonstrated the application value of TGS-based comprehensive analysis of thalassemia alleles in the detection of rare thalassemia gene variants.

Case Report: The third-generation sequencing confirmed a novel 7.2 Kb deletion at ß-globin gene in a patient with rare ß-thalassemia.

Background: Thalassemia was the most common monogenic diseases worldwide, which was caused by mutations, deletions or duplications in human globin genes which disturbed the synthesis balance between α- and ß-globin chains of hemoglobin. There were many classics methods to diagnose thalassemia, but all of them had limitations. Although variations in the human ß-globin gene cluster were mainly point mutations, novel large deletions had been described in recent years along with the development of DNA sequencing technology. Case report: We present a case of 32-year-old male with abnormal hematological results. However, 23 genotypes of the most common thalassemia were not detected by two independent conventional platforms. Finally, using multiplex ligation-dependent probe amplification (MLPA), third-generation sequencing (TGS) and Gap PCR detection methods, we first confirmed the case with a novel 7.2 Kb deletion (Chr11:5222800-5230034, hg38) located at HBB gene. Conclusion: Our results showed that TGS technology was a powerful tool for thalassemia breakpoint detection, had promising potentiality in genetic screening of novel thalassemia, especially for the novel deletions in globin genes.

A novel rearrangement of the α-globin gene cluster containing both the -α3.7 and ααααanti4.2 crossover junctions in a Chinese family.

BACKGROUND: Thalassemia is one of the most common hemoglobinopathies. Thalassemia is mainly caused by the loss and/or deficiency of one or more globin chains in hemoglobin. The copy number variant (CNV) of α-globin gene is one of the important factors affecting the clinical phenotype of ß-thalassemia. The precise detection for this type of variation is needed. METHODS: Peripheral blood of a 33-year-old man and his family members were collected. Complete blood counts and serum iron levels were measured for participants. Genomic DNA was extracted from all family members. Routine genetic analysis of thalassemia was performed to determine the genotype. Additional PCR-electrophoresis and Multiplex ligation dependent probe amplification (MLPA) were conducted. Single-molecule real-time technology(SMRT) was then performed as a validation assay and further characterization of the variant for family members. RESULTS: PCR-electrophoresis and MLPA found a new variant, but the exact genotype could not be determined. At last, SMRT identified the new variant as a rearrangement of the α-globin gene cluster named αHKαα (NC_000016.9:g.169818_174075dup169818_174075dup173302_177105del), which contained both the -α3.7 and ααααanti4.2 crossover junctions. Carriers of the novel CNV show normal clinical phenotype according to the hematological results. CONCLUSION: We have identified an unreported CNV (αHKαα) in α-globin gene cluster. The novel CNV not only demonstrates the accuracy and efficiency of our combining strategy in detecting unknown CNVs, but also enriched the variant spectrum of thalassemia.

A novel 15.8 kb deletion α-thalassemia confirmed by long-read single-molecule real-time sequencing: Hematological phenotypes and molecular characterization.

BACKGROUND: Thalassemia is the most frequent recessive Mendelian inherited monogenic disease worldwide, and is characterized by the impaired synthesis of globin chains due to disease-causing variants in α- or ß-globin genes. There are many conventional methods to diagnose thalassemia but all of them have limitations. CASE REPORT: We present the case of a 37-year-old female with abnormal values of routine hematological indices who was admitted for genetic screening of thalassemia. Genomic DNA was extracted and used for genetic assays covering the known and potential novel genotypes in HBA and HBB genes using a suspension-array system, gap-polymerase chain reaction (Gap-PCR), PCR-reverse dot blot (PCR-RDB) and multiplex ligation-dependent probe amplification (MLPA). Finally, using long-read single-molecule real-time (SMRT) sequencing, we first confirmed the case with a novel 15.8 kb deletion located in the HBA gene (Chr16:163886-179768, GRch38/hg38). CONCLUSIONS: Our results showed that long-read SMRT sequencing has great advantages in the detection of rare α-globin gene variants. This study may provide a reference protocol for the use of long-read SMRT sequencing for the detection of known and potential novel genotypes of thalassemia in the population and improve the accuracy of genetic counseling and prenatal diagnosis.

Analysis of rare thalassemia genetic variants based on third-generation sequencing.

Thalassemia is a group of common hereditary anemias that cause significant morbidity and mortality worldwide. However, precisely diagnosing thalassemia, especially rare thalassemia variants, is still challenging. Long-range PCR and long-molecule sequencing on the PacBio Sequel II platform utilized in this study could cover the entire HBA1, HBA2 and HBB genes, enabling the diagnosis of most of the common and rare types of thalassemia variants. In this study, 100 cases of suspected thalassemia were subjected to traditional thalassemia testing and third-generation sequencing for thalassemia genetic diagnosis. Compared with traditional diagnostic methods, an additional 10 cases of rare clinically significant variants, including 3 cases of structure variants and 7 cases of single nucleotide variations (SNVs) were identified, of which a case with - α3.7 subtype III (- α3.7III) was first identified and validated in the Chinese population. Other rare variants of 11.1 kb deletions (- 11.1/αα), triplicate α-globin genes (aaa3.7/αα) and rare SNVs have also been thoroughly detected. The results showed that rare thalassemia variants are not rare but have been misdiagnosed by conventional methods. The results further validated third-generation sequencing as a promising method for rare thalassemia genetic testing.

Change point detection for COVID-19 excess deaths in Belgium.

Emerging at the end of 2019, COVID-19 has become a public health threat to people worldwide. Apart from deaths with a positive COVID-19 test, many others have died from causes indirectly related to COVID-19. Therefore, the COVID-19 confirmed deaths underestimate the influence of the pandemic on society; instead, the measure of 'excess deaths' is a more objective and comparable way to assess the scale of the epidemic and formulate lessons. One common practical issue in analysing the impact of COVID-19 is to determine the 'pre-COVID-19' period and the 'post-COVID-19' period. We apply a change point detection method to identify any change points using excess deaths in Belgium.

The value of single-molecule real-time technology in the diagnosis of rare thalassemia variants and analysis of phenotype-genotype correlation.

To compare single-molecule real-time technology (SMRT) and conventional genetic diagnostic technology of rare types of thalassemia mutations, and to analyze the molecular characteristics and phenotypes of rare thalassemia gene variants, we used 434 cases with positive hematology screening as the cohort, then used SMRT technology and conventional gene diagnosis technology [(Gap-PCR, multiple ligation probe amplification technology (MLPA), PCR-reverse dot blot (RDB)] for thalassemia gene screening. Among the 434 enrolled cases, conventional technology identified 318 patients with variants (73.27%) and 116 patients without variants (26.73%), SMRT identified 361 patients with variants (83.18%), and 73 patients without variants (16.82%). The positive detection rate of SMRT was 9.91% higher than conventional technology. Combination of the two methods identified 485 positive alleles among 49 types of variant. The genotypes of 354 cases were concordant between the two methods, while 80 cases were discordant. Among the 80 cases, 76 cases had variants only identified in SMRT method, 3 cases had variants only identified in conventional method, and 1 false positive result by the traditional PCR detection technology. Except the three variants in HS40 and HBG1-HBG2 loci, which was beyond the design of SMRT method in this study, all the other discordant variants identified by SMRT were validated by further Sanger sequencing or MLPA. The hematological phenotypic parameters of 80 discordant cases were also analyzed. SMRT technology increased the positive detection rate of thalassemia genes, and detected rare thalassemia cases with variable phenotypes, which had great significance for clinical thalassemia gene screening.