Dataset Information

Accurate genotype diagnosis of Hong Kongαα thalassemia based on third-generation sequencing.

ABSTRACT:

Background

The Hong Kongαα (HKαα) allele is a complex structural rearrangement of the α-globin gene containing -α^3.7 and ααα^{anti 4.2} crossover junctions. Clinically, individuals carrying the HKαα allele are often misdiagnosed or missed using conventional thalassemia gene detection technology. This study aims to identify and validate different HKαα thalassemia subtypes using third-generation sequencing (TGS) technology.

Methods

Between January 2015 and June 2021, 32 patients suspected of having HKαα thalassemia were included in this study. Genomic DNA was extracted, and gap-polymerase chain reaction (PCR), two-round nested PCR, multiplex ligation-dependent probe amplification (MLPA), and TGS were used for thalassemia gene detection.

Results

The results of HKαα/αα and HKαα/-α^3.7 were similar to -α^3.7/αα using the gap-PCR method. Two-round nested PCR could be used to verify the HKαα gene, but could not distinguish the subtypes of HKαα thalassemia. The MLPA assay was used to detect the change in the copy number of the α-globin gene, but it could not determine whether -α^3.7 and ααα^{anti 4.2} were in cis or in trans. Long-read TGS technology could accurately detect the HKαα allele and distinguish the genotypes of HKαα/αα, HKαα/-α^3.7, HKαα/-α^4.2, and HKαα/--^SEA without pedigree analysis. The contiguous sequence of the HKαα allele was detected using the TGS approach. This study also demonstrated that individuals with HKαα/αα and β^N/β^N genotypes tended to have normal hematological phenotypes.

Conclusions

Long-read TGS is a reliable and efficient approach for accurate detection of HKαα thalassemia, which can be widely used in clinical practice. Accurate molecular diagnosis of HKαα thalassemia will benefit clinical genetic counseling and prenatal diagnosis.

SUBMITTER: Li J

PROVIDER: S-EPMC9652573 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Publications

Accurate genotype diagnosis of Hong Kongαα thalassemia based on third-generation sequencing.

Li Jun J Ye Guimei G Zeng Dan D Tian Baodong B Wang Wenjuan W Feng Qiao Q Zhu Chunjiang C

Annals of translational medicine 20221001 20

<h4>Background</h4>The Hong Kongαα (HKαα) allele is a complex structural rearrangement of the α-globin gene containing -α<sup>3.7</sup> and ααα<sup>anti 4.2</sup> crossover junctions. Clinically, individuals carrying the HKαα allele are often misdiagnosed or missed using conventional thalassemia gene detection technology. This study aims to identify and validate different HKαα thalassemia subtypes using third-generation sequencing (TGS) technology.<h4>Methods</h4>Between January 2015 and June 20 ...[more]

PMID: 36388814

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Project description:BackgroundThalassemia is one of the most common monogenic hemolytic disorders in the world. Hong Kongαα (HKαα) thalassemia was initially found among the people of southern China. Because of the complexity of genetic changes in HKαα thalassemia, we lack a precise sequence analysis of the HKαα allele. Here we aim to detect the specific genotype and trace the law of inheritance of this rare genotype.MethodsWe recruited an unprecedented huge pedigree containing 11 individuals carrying the HKαα thalassemia gene and 4 nongenetic-related patients suffering from HKαα from south China. Regular hematological analysis and routine genetic screening were performed on the pedigree and two-round nested PCR (polymerase chain reaction) for HKαα thalassemia were performed on each individual. The first-generation gene sequencing was performed on six individuals, including four nongenetic-related patients.ResultWe found that five family members were positive for the HKαα allele. Patients Ⅱ-2, Ⅲ-1, and Ⅱ-3 with only HKαα/--SEA or HKαα/-α4.2 presented with α-thalassemia minor trait. Ⅰ-1, the carrier of both HKαα/-α3.7 and β41-42 /βN , showed a typical β-thalassemia trait. Fetus with genotype HKαα/-α4.2 alone was not likely to suffer from any deleterious effects after birth. The whole sequence of HKαα allele revealed that HKαα alleles in the six patients shared a high similarity, implying that all HKαα alleles are likely from the same ancestor. Moreover, pedigree and sequencing analyses demonstrated that the HKαα allele contained αααanti4.2 mutation, -α3.7 mutation, and a fragment from α-hemoglobin gene; thus, the composition and formation of HKαα allele was revealed. Finally, the high similarity and composition of HKαα alleles implies that once HKαα formed, αααanti4.2 and -α3.7 mutations tended to be a fusion gene and quite impossible to be inherited separately.ConclusionThe two-round nested PCR is an effective method to detect HKαα allele. Besides, our study for the first time revealed the sequence of the HKαα allele, the evidence of the same ancestor with HKαα thalassemia and enriched the composition as well as the formation mechanism of HKαα allele, and immediately opened up novel potential diagnosis and prenatal counseling for HKαα thalassemia.

Project description:Infections of the ascitic fluid are serious conditions that require rapid diagnosis and treatment. Ascites is often accompanied by other critical pathologies such as gastrointestinal bleeding and bowel perforation, and infection increases the risk of mortality in intensive care patients. Owing to a relatively low success rate of conventional culture methods in identifying the responsible pathogens, new methods may be helpful to guide antimicrobial therapy and to refine empirical regimens. Here, we aim to assess outcomes and to identify responsible pathogens in ascitic fluid infections, in order to improve patients' care and to guide empirical therapy. Between October 2019 and March 2021, we prospectively collected 50 ascitic fluid samples from ICU patients with suspected infection. Beside standard culture-based microbiology methods, excess fluid underwent DNA isolation and was analyzed by next- and third-generation sequencing (NGS) methods. NGS-based methods had higher sensitivity in detecting additional pathogenic bacteria such as E. faecalis and Klebsiella in 33 out of 50 (66%) ascitic fluid samples compared with culture-based methods (26%). Anaerobic bacteria were especially identified by sequencing-based methods in 28 samples (56%), in comparison with only three samples in culture. Analysis of clinical data showed a correlation between sequencing results and various clinical parameters such as peritonitis and hospitalization outcomes. Our results show that, in ascitic fluid infections, NGS-based methods have a higher sensitivity for the identification of clinically relevant pathogens than standard microbiological culture diagnostics, especially in detecting hard-to-culture anaerobic bacteria. Patients with such infections may benefit from the use of NGS methods by the possibility of earlier and better targeted antimicrobial therapy, which has the potential to lower the high morbidity and mortality in critically ill patients with ascitic bacterial infection.

Dataset Information

Accurate genotype diagnosis of Hong Kongαα thalassemia based on third-generation sequencing.

Background

Methods

Results

Conclusions

Publications

Accurate genotype diagnosis of Hong Kongαα thalassemia based on third-generation sequencing.

Similar Datasets

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