Thalassemia including α-thalassemia and β thalassemia is among the most common hereditary disorders in the world [1]. Compared to β thalassemia, α-thalassemia occurs at a high frequency due to decreased or absent synthesis of α-globin chains. In southern China, the carrier rate of α-thalassemia ranges from 5-15% [2]. The level of α-globin chains in compound heterozygotes and homozygotes significantly decreases, which causes HbH disease or Hb Bart's hydrops. Hb Bart’s (c4) hydrops fetalis is often associated with severe obstetric complications for the mother and perinatal death [3]. Patients with HbH disease will replay on transfusion, thus their quality of life and life expectancy are suboptimal and the economic burden is heavy [4]. Carrier screening has been proven to be an effective method to prevent the birth of neonates with severe thalassemia [5]. Although several technologies such as MLPA and Sanger sequencing have been performed together, approximately 56,000 babies are even born with major thalassemia [6]. The existence of complex variants including repetitive regions and homology can affect the accuracy of molecular and genetic analysis methods. In recent years, third-generation sequencing (TGS) has been reported to be useful for complex structural variations due to the advantage of longer read length [7, 8]. In this report, we first describe the case with a special deletion of the α-globin gene cluster identified using third-generation sequencing, which can provide the potential diagnosis method for rare complex genotypes.
The proband was a 32-year-old male originated from Panyu City, Guangdong Province in southern China. He and his wife did premarital screening tests for thalassemia, and both were screened positive for α-thalassemia based on the results of red blood cell parameters and hemoglobin analyses (Table 1). The α-globin genotyping with MeltPro HBA assays (Xiamen Zeesan Biotech Co., Ltd., Xiamen, Fujian, China) which can simultaneously detects four common deletional α-thalassemia (--SEA, --THAI, –α3.7 and –α4.2) and three nondeletional α-thalassemia (HBA2: c.427T>C, HBA2: c.369C>G, HBA2: c.377T>C), revealed that the female was a --SEA deletion carrier. However, the husband was detected negative using this approach, and later the result of Gap-PCR to detect the four common α-thalassemia deletions was also negative. In order to exclude rare α-thalassemia, both Sanger sequencing and SALSA MLPA probemix P140 HBA protocol (MRC-Holland, Amsterdam, Netherlands) were used to detect single nucleotide variations (SNVs) of the entire α-globin genes (α1 and α2) and copy number variations (CNVs) within α-globin gene cluster respectively in this patient. While Sanger sequencing of the α-globin gene was negative, a heterozygous deletion across 21 probes was detected by MLPA, similar to --SEA deletion (Fig. 1A), suggesting the presence of an unknown deletion in the man. In order to get more details about the breakpoints, we designed five MLPA probes to cover the related regions (Fig. 1B, Table S1). With the information provided by these probes, we designed special primers for longer PCR to cover the whole deletion (Table 1). As a result, a fragment of approximately 7kb was amplificated (Fig. 1C). We then used three primers for sequencing the fragment, and successfully targeted the breakpoints (Fig. 1D, E). Surprisingly, we found that the fragment in the breakpoint is located at α-globin gene cluster (Chr16:154445–154538, GRch38/hg38) and out of the amplification range (Chr16:161172–184467, GRch38/hg38), indicating the existence of homologous recombination. It was difficult to obtain the information due to GC-rich regions using the above methods. Thus, to identify this unknown deletion, we performed TGS (Pacific Biosciences Inc., Menlo Park, USA) and successfully determined the novel deletion (hg38, Chr16:165236-182113) (Fig 2A). Clinical and molecular information of the proband and his family members were described in Table 1.
α-thalassaemia is caused by mutations or deletions of the α-globin genes. Approximately 90% of the mutations is the deletional type because the HBA1 gene has strong homology to HBA2 gene. MeltPro HBA assays and Gap-PCR for four common deletions are used in conventional thalassemia genetic analysis [9]. The different results have been reported due to the difference in the detection principle of those two methods by our team [7]. MLPA is often performed to validate the unknown CNVs in the α or β gene globin cluster, but the exact location could not be clarified. Sanger sequencing is considered as an effective method for single nucleotide variations (SNVs), but it is not appropriate for CNVs. Most importantly, high quality reads of this method were only up to 1kb [10], so it is difficult to provide more complete information for long fragments. For third-generation sequencing, longer reads and lacking amplification biases permit sequencing through repetitive elements and high GC-rich regions. The existence of high GC-rich regions and highly homologous sequences is the key characteristics of α-globin gene cluster, indicating that TGS is very suitable for α-thalassemia [8, 11, 12].
The SALSA MLPA probemix P140 HBA protocol has been used for detecting rare α-thalassemia carriers for many years. In this case, the MLPA results of both the proband’s father and proband showed --SEA deletion based on the ratio and location of probes according to the protocol while no abnormalities were observed using MeltPro HBA assays and Gap-PCR. Low MCV and MCH value suggested the existence of rare thalassemia. It gave clinicians a great confusion. The distance between MLPA probes is too long to design primers for confirming the precise localization of deletion breakpoints. In order to discriminate the rare deletional thalassemia from --SEA deletion, we designed special probes and obtained accurate information about downstream breakpoint. Unexpectedly, we detected a fragment ranging from 153226 to 154538(GRch38/hg38) near upstream breakpoint, which was out of the amplified fragment’s range using these primers (chr16: 161172 to 184467). Subsequently, the homology sequence (chr16:163816–164943, GRch38/hg38) was found in UCSC database (UCSC Genome Browser Home), suggesting the existence of the homologous recombination event. However, due to the limitation of Sanger sequencing that it could not detect the existence of complex structures such as poly A and GC-rich regions, we could not identify the detailed structure of the fragment. Fortunately, the TGS provided a complete genetic blueprint of this rare genotype and presented the advantages of accurate screening of thalassemia carriers. Based on the TGS results, we hypothesize that the misalignment between those two homologous fragments and unequal crossover occurred during the meiosis phase of the gametogenesis process (Fig 2B, C). The breakpoint of the upstream sequence was similar to that of --SEA deletion, which impacted the forward primer binding in Gap-PCR and the mutated probe binding in MeltPro HBA assays.
As we all know, MLPA has been used for detecting CNV in α or β globin gene cluster for many years, but there are several reasons limit its usage and accuracy. First, the number of probes in the commercial kits is limited so more special probes need to be designed in order to obtain enough information. Second, designing probes and optimizing the reaction system are sometimes difficult to perform in the laboratory, Third, the cost of synthesis of probs is relatively high, ~ $12 USD per probe. Notably, after MLPA analysis, we still need to use PCR and Sanger sequencing to obtain the accurate information of sequences. Thus, a better way to detect the gene mutations in α-thalassemia can be used. The TGS including Single-molecule real-time (SMRT) sequencing by Pacific Biosciences (PacBio), nanopore sequencing by Oxford Nanopore technologies, and long-read SMRT sequencing analysis has been confirmed very suitable for genetic testing of thalassemia [13], with modest cost which is less than $20 per sample. More specifically, the SMRT technology used in the genetic diagnosis of thalassemia has a wider detection spectrum, and has been confirmed to be better used for complicated genotypes of thalassemia compared to traditional analytic methods such as MLPA, PCR, and Sanger sequencing. The case we reported is a classic example of its application.
In conclusion, in this case, we reported a novel deletion in the α-globin cluster using different methods. The result of TGS can be identified by synthesizing of MLPA and longer PCR. Based on our knowledge, this is the first report on the novel deletion using TGS method. This research fully presented the advantages of TGS and enriched the database of thalassemia variants. Moreover, based on the results, we provided detailed and accurate genetic counseling to the family members. The wife will also be performed prenatal diagnosis when she gets pregnant. Our study gives some novel ideas for exploring the diagnostic methods of complex variants in the α-thalassemia gene.