Detection Of Rare Thalassemia Mutation Using Long-Read Single-Molecule Real-Time Sequencing

RATIONALE PURPOSEThalassaemia is an autosomal recessive disorder that is widely distributed worldwide.The current effective treatment for severe thalassaemia is bone marrow transplantation,but the cost of daily treatment is high.The birth of children with severe thalassaemia brings a heavy burden to the country,society and family,so prenatal screening and prenatal diagnosis of fetuses with severe thalassaemia are particularly important.The condition for prenatal diagnosis is that the genotype of the proband or his parents must be clear.At present,there are many molecular diagnostic detection methods for thalassaemia in China,one is the technology for the detection of common types of thalassaemia genes,including cleft polymerase chain reaction,reverse spot hybridization technology,PCR-based multi-color dissolution curve analysis,etc.,and the other is to detect a wider range of gene mutation profiles,including multiple-ligation probe amplification technology,generation sequencing,etc.Although the aforementioned methods can detect some rare thalassaemia,due to the limitations of various methods,multiple methods are still needed to be combined to detect,and rare thalassaemia caused by rare complex structural variations cannot be clearly diagnosed or missed,resulting in the occurrence of severe thalassaemia,so it needs to attract great clinical attention.How to quickly and effectively detect rare type of thalassaemia is a problem that needs to be often faced in clinical practice.Third-generation sequencing technology is an emerging technology in recent years,including single molecule real time sequencing technology（SMRT）and nanopore sequencing technology,the former is more mature.Third-generation sequencing technology is now used in genetic testing,but thalassemia testing has been less reported.PURPOSE1.Nine rare thalassemia families have been reported.2.In this study,single-molecule real-time sequencing technology was used to solve the clinical cases of rare thalassemia that are difficult to diagnose by traditional PCR technology,and the feasibility of third-generation sequencing technology in the clinical diagnosis of rare thalassemia was explored.METHODS1.From January 2021 to October 2022,9 rare cases of thalassaemia with suspected phenotype suspected of thalassaemia but the molecular diagnosis results of traditional PCR detection technology did not match or could not detect results were collected.2.Blood samples from nine proband suspected rare thalassemia and members of their families were sent for third-generation sequencing.3.After the sequencing results were returned,they were analyzed,and other molecular biology methods were used to verify and perform family analysis,and whether the results were different from those of traditional thalassaemia molecular diagnostic techniques.Combined with clinical medical records and other examination results,the accuracy of third-generation sequencing technology in detecting rare thalassemia was analyzed,and the pathogenesis of rare thalassemia was explored.4.Summarize the advantages and disadvantages of third-generation sequencing technology in the diagnosis of rare thalassaemia,and provide laboratory basis for selecting the appropriate diagnostic pathway in clinical practice.RESULTSE1.The genotypes of 9 rare thalassemia samples were identified,includingɑ^762bpɑ/ɑɑ,gene insertion of 342bp,ɑ^QSɑ/ɑ^Fusionɑ,ɑɑɑ^anti3.7/HKɑɑ,--^SEA/ɑɑɑ,--^SEA/ɑ1ɑ1,-ɑ^3.7/ɑ1ɑ1,ɑɑ^anti3.7ɑ^anti4.2ɑ/ɑɑ,ɑɑɑɑ/ɑɑ,and provided clinical hematological phenotypic information.2.Four genotype types were reported for the first time,includingɑ^762bpɑ/ɑɑ,,ɑ^QSɑ/ɑ^Fusionɑ,ɑɑɑ^anti3.7/HKɑɑ,ɑɑ^anti3.7ɑ^anti4.2ɑ/ɑɑ.3.The production mechanism of the above rare thalassemia samples was preliminarily determined,including 1 rare deletion,1 rare insertion,1 rare replicate,2 gene fusions,2 complex triplets and 2 gene exchange structure rearrangements.CONCLUSION1.For rare thalassaemia,even the combined use of multiple traditional genetic diagnostic techniques（Gap-PCR,RDB,MMCA,MLPA,etc.）can lead to missed diagnosis and misdiagnosis of thalassaemia,which has great limitations.2.Compared with traditional genetic testing technology,SMRT technology is more suitable for rare thalassaemia gene detection caused by complex structural variation,and can clearly identify cis-and trans.3.Third-generation sequencing based on clinical phenotype and family analysis can be used as the preferred laboratory detection method for clinical diagnosis of rare thalassaemia and analysis of pathogenicity.4.Clarify the limitations of traditional detection methods in detecting rare thalassaemia,especially those caused by complex structural variations,and provide a preliminary clinical diagnosis path for the discovery and diagnosis of rare thalassaemia caused by complex structural variations.