Thalassemia is one of the most common single gene inheritance diseases in the world. It is caused by the interruption of α- or β-globin chain synthesis, leading to an imbalance in the ratio of α/non-α chains. -α3.7, -α4.2, and --SEA are the most common α-thalassemia (α-thal) deletions found in China. The overall prevalence of --SEA, -α3.7, and -α4.2 was 68.06%, 25.0%, and 2.78%, respectively, in Chengdu, Sichuan, China. In 2005, Wang et al found a rare rearrangement of the α-globin gene cluster, HongKongαα (HKαα) allele, which contains both -α3.7 and αααanti-4.2 crossover junctions. The -α3.7 and αααanti-4.2 fragments are located on the same chromosome as HKαα, whereas on two chromosomes, it is -α3.7/αααanti-4.2. However, both HKαα and -α3.7/αααanti-4.2 are misdiagnosed as -α3.7/αα by the current conventional thalassemia detection methods.
The HKαα allele contains neither deletion nor triplication, and carriers of this allele are unlikely to suffer any deleterious effects. Moreover, there were no differences between HKαα/--SEA and -α3.7/αα in erythrocyte parameters or hemoglobin electrophoresis results.[4,5] Once HKαα is misdiagnosed as the -α3.7 deletion, and the patient's spouse is --SEA/αα, while unnecessary amniocentesis is required for a clear diagnosis, increasing the risk of abortion and economic burden, causing psychological stress.
The clinical manifestations of -α3.7/αααanti-4.2 are similar to HKαα in theory. However, as the genotype is rare, the number of reports is small. When the patient's spouse is a carrier of β-thalassemia (β-thal), their offspring have a 1/4 probability of αααanti-4.2 and β-thal coinheritance. ααα triploid aggravates the imbalance of α/β due to the increase in the α-globin chain, which aggravates the clinical manifestation of β-thalassemia.[7–11] Therefore, when -α3.7/αααanti-4.2 is misdiagnosed as -α3.7/αα, the genetic probability of moderate to severe thalassemia may be misjudged by clinicians, and may exacerbate the birth of anemic children.
At present, routine testing for thalassemia cannot detect HKαα and -α3.7/αααanti-4.2. Gap-polymerase chain reaction (Gap-PCR) and reverse dot blot (RDB) assays are widely used to detect the common α-thal and β-thal mutations.[12,13] The HKαα allele and some additional genotypes (HKαα/--SEA, HKαα/-α4.2, and HKαα/αCSα) formed by HKαα can be detected by multicolor melting curve analysis with real-time PCR. However, none of the three methods can detect triplication of the α-globin gene. Nested PCR is a cost-effective way to detect the HKαα allele, but it cannot identify additional genotypes. Sequencing is well suited to the detection of small sequence changes and is less well adapted for detecting structural variants. Multiplex ligation-dependent probe amplification (MLPA) is a useful technique for detecting and identifying copy number variation (deletions/duplications) in any region of the genome.[16,17] This technique has been applied to detect the HBA and HBB gene.[18–21] Xie et al detected HKαα/αα with MLPA. However, when the balanced translocation of chromosomes coexists in samples, the results of MLPA are incorrect. Some genotypes, such as -α4.2/αααanti-4.2 and -α3.7/αααanti-3.7, may be diagnosed in normal subjects by this technology.
Therefore, nested PCR combined with MLPA was used to analyze the genotypes of αααanti-4.2 and HKαα in patients with -α3.7 deletion thalassemia, and to ascertain whether it coexists with other genotypes of thalassemia.
The study was followed the principles of Declaration of Helsinki and approved by the Institutional Review Committee of Hospital of University of Electronic Science and Technology of China and Sichuan Provincial People's Hospital (No. 2019-1). Informed consent was obtained from each patient and their family members before genetic testing was performed.
Samples and hematologic analysis
A total of 5488 peripheral blood samples for thalassemia testing were collected from July 2017 to October 2019 in Sichuan Provincial People's Hospital, China. Of these, 1461 (26.62%) were male and 4027 (74.38%) female. The patients were in the age range of 2 month-60 years (27.72±10.53 years). The hematological parameters of the samples were determined by an automatic analyzer XN-10 (Sysmex Corporation, Kobe, Japan). A capillary electrophoresis device (Capillarys; Sebia, Montpellier, France) was used for hemoglobin electrophoresis analysis.
Analysis of common thalassemia mutations by Gap-PCR and RDB
Genomic DNA was extracted from peripheral blood. Single-tube multiplex Gap-PCR was used to test the four α-globin gene deletion (-α3.7, -α4.2, --SEA, and --THAI) (Shenzhen Yishengtang Biological Enterprise Co., Ltd, Shenzhen, China). All DNA samples with the -α3.7 deletion were collected.
The RDB assay was used for three common non-deletional α-thal mutations (Hb Constant Spring [Hb CS] HBA2: c.427T>C, Hb Quong Sze [Hb QS] HBA2: c.377T>C, and Hb Westmead [Hb WS] HBA2: c.369122C>G] and 17 known Chinese β-thal mutations (Shenzhen Yishengtang Biological Enterprise Co., Ltd).
Analysis of the genotypes of αααanti-4.2 and HKαα allele
The αααanti-4.2 junction was assessed by the anti-4.2 multiplex-PCR assay for samples diagnosed as -α3.7. All primers were used to detect the HKαα allele [Table 1].[6,13] Each 20 μL reaction contained the following: 10 ng/μL DNA, 1× GC Buffer I, 0.4 mmol/L dNTP, 0.3 μmol/L anti-4.2-F, 0.3 μmol/L anti-4.2-R, 0.15 μmol/L LIS1-2.5-F, 0.15 μmol/L LIS1-2.5-R, 0.05 IU/μL Takara LA-Taq (Takara Bio, Shiga, Japan). Double-distilled water was added to a volume of 20 μL. The PCR procedure was as follows: 95° C for 1 min initially, followed by 35 cycles of 94° C for 30 s and 68° C for 3 min, then a final 3 min at 68° C.
Two-round nested PCR was carried out in samples with both -α3.7 and αααanti-4.2 [Figure 1]. The first-round PCR amplified a DNA segment from X1 to Z1 boxes and a DNA segment for internal quality control. Each 20-μL reaction contained the following: 10 ng/μL DNA, 1× GC Buffer I, 0.4 mmol/L dNTP, 0.4 μmol/L L-anti-4.2-F, 0.4 μmol/L L-α3.7-R, 0.1 μmol/L LIS1-2.5-F, 0.1 μmol/L LIS1-2.5-R, 0.05 IU/μL Takara LA-Taq, and double-distilled water was added to a volume of 20 μL. The PCR procedure was as follows: 95° C for 1 min initially, followed by 35 cycles of 94° C for 30 s and 68° C for 5 min, then a final 3 min at 68° C.
The second-round PCR was used to amplify the αααanti-4.2 duplication. For general amplification success, the LIS1-2.0 primers were also used as the internal quality control. The primers used in the nested PCR except for LIS1-2.5 and LIS1-2.0 primers are labeled in Figure 1. Each 20-μL reaction contained the following: 1 μL of the first-round PCR products diluted 1600 times as template, 0.4 mmol/L MgCL2, 1× EX Buffer (Mg2+ free), 0.4 mmol/L dNTP, 0.3 μmol/L AT4.2-F, 0.3 μmol/L AT4.2-R, 0.05 μmol/L LIS1-2.0-F, 0.05 μmol/L LIS1-2.0-R, and 0.05 IU/μL Takara EX Taq, and double-distilled water was added to a volume of 20 μL. The PCR procedure was as follows:95°C for 1 min initially, followed by 35 cycles of 95°C for 30 s, 56°C for 45 s, and 72°C for 60 s, then a final 3 min at 72°C.
Assessment of the copy number of the HBA gene by MLPA
For samples carrying both -α3.7 and αααanti-4.2, MLPA analysis was carried out using the SALSA MLPA KIT HBA140-C1 kit (MRC-Holland, Amsterdam, The Netherlands) according to the manufacturer's instructions. Three wild-type subjects were collected as references, and three common α-thal deletions (-α3.7/αα, -α4.2/αα, and --SEA/αα) were used as positive controls of the α-globin gene cluster for MLPA. In short, the genomic DNA was denatured and hybridized with SALS-MLPA probes specific to the α-globin gene cluster. After ligation, PCR was performed using primers specific to the probes. The amplification results were analyzed on an ABI PRISM 3730 (Applied Biosystems, Foster City, CA, USA) Genetic Analyzer. The data were analyzed by coffalyser.net (MRC-Holland).
The statistical software SPSS version 17.0 (SPSS Inc., Chicago, IL, USA) was applied for statistical analysis. Fisher's exact test was used to detect a statistically significant difference between the Gap-PCR and Gap-PCR combined with nested PCR and MLPA in detecting HKαα allele. Statistical significance was set as P < 0.05.
Identification of thalassemia mutations by Gap-PCR and RDB assays
Total 2544 cases were identified as thalassemia in 5488 peripheral blood samples. The results showed that α, β, and αβ compound thalassemia were identified in 1190 (46.78%), 1286 (50.55%), and 68 (2.67%) cases, respectively. A total of 227 samples from thalassemia patients were identified as -α3.7/αα by Gap-PCR. The other two patients were suspected to be HKαα carriers due to the presence of three bands (2.0, 1.7, and 1.2/1.4 kb, respectively) in the electrophoresis study.
Analysis of the genotypes of αααanti-4.2 and the HKαα allele
Of the 229 patients who were identified as -α3.7/αα or suspected to be HKαα carriers, 20 patients were identified as HKαα carriers, and one patient was identified as a -α3.7/αααanti-4.2 carrier by two-round nested PCR and MLPA, including 15 patients of HKαα/αα, three patients of HKαα/αα and β-thalassemia coinheritance, one patient with HKαα/--SEA, one patient with HKαα/-α4.2 and β-thalassemia coinheritance, and one patient with -α3.7/αααanti-4.2 and β-thalassemia coinheritance. The phenotypes and genotypes of the cases carrying the -α3.7 deletion and αααanti-4.2 duplication are summarized in Table 2. The interpretation of the PCR and MLPA results is shown in Table 3.
Comparison between Gap-PCR and the combination of Gap-PCR, nested PCR and MLPA in detecting HKαα allele
There was a significantly difference between Gap-PCR and Gap-PCR combined with nested PCR and MLPA in detecting HKαα (P < 0.05) [Table 4]. Of the 229 patients, two patients were suspected of carrying HKαα by Gap-PCR. Gap-PCR combined with nested PCR and MLPA found that 21 patients were HKαα carriers or -α3.7/αααanti-4.2. The error rate of diagnosis was 9.17% (21/229) by Gap-PCR in -α3.7 deletion.
In a clinical test, -α3.7/αα, HKαα/αα, HKαα/-α3.7, HKαα/αααanti-4.2, HKαα/αααanti-3.7, and -α3.7/αααanti-4.2 may be misdiagnosed as -α3.7/αα. Different genotypes may lead to different clinical phenotypes. According to the literature, there is a considerable difference in the carrier frequency of HKαα in different geographical populations, and the carrying rate is about 0.07% to 2.27%.[5,24] In this study, of the 229 patients, 20 patients were identified as HKαα carriers, and one patient was identified as -α3.7/αααanti-4.2 by two-round nested PCR and MLPA. The frequency of HKαα allele was as high as 8.81% among the -α3.7 carriers. The error rate of diagnosis is 9.17% (21/229) by Gap-PCR in -α3.7 deletion. Thus, to obtain a more accurate diagnosis and treatment, it was necessary to distinguish -α3.7 from HKαα and -α3.7/αααanti-4.2.
In this study, when people were diagnosed as carriers of -α3.7, anti-4.2 multiplex-PCR was adopted to determine whether to continue supplementary experiments. However, HKαα/αα, HKαα/αααanti-4.2, HKαα/αααanti-3.7, HKαα/HKαα, and HKαα/-α3.7 could not be discerned from each other by Gap-PCR, anti-4.2 multiplex-PCR or nested PCR [Table 3]. MLPA analysis was used to determine the number of copies of the HBA gene. However, when there are balanced translocations of chromosomes in specimens, they could not be correctly assessed by MLPA [Figure 2C]. In addition, MLPA is unable to determine whether deletions and duplications are located on the same chromosome. Thus, HKαα/αα could not be distinguished from -α3.7 /αααanti-4.2 [Figure 2A]. Therefore, in this study, nested PCR was performed to detect the HKαα allele, and MLPA analysis was not only used to ensure the results of the nested PCR but also to find extra deletions or duplications. Thus, these two techniques could aid and verify each other [Figure 2].
All parameters were normal except for patients 2, 7, 10, 11, 12, 16, and 19. They might have been caused by the coinheritance of β-thal, -α4.2, or --SEA deletions. The other patients were HKαα heterozygotes, of which patient 7 showed obvious small-cell hypochromic anemia, which was confirmed by clinical analysis and combined with iron deficiency anemia. The hematological parameters of the other patients were almost normal, which was consistent with the results of Wang et al and Wu et al. Patient 16 is -α3.7/αααanti-4.2 combined with βIVS-2-654/βN, which is rarer than HKαα. After testing, the patient's spouse was found to have a normal genotype, and their offspring have a 1/4 probability of -α3.7/αα complex βN/βN, 1/4 probability of -α3.7/αα complex βN/βIVS-2-654, 1/4 probability of αα/αααanti4.2 complex βN/βN, and 1/4 probability of αα/αααanti-4.2 complex βN/βIVS-2-654. The αα/αααanti4.2 complex βN/βIVS-2-654 genotype can aggravate the clinical manifestations of β-thal. Therefore, genetic counseling should be carried out during pregnancy.
At present, there is no gold standard for testing the accurate genotypes of HKαα carriers as a reference. Thus we did not determine precise rates of this combination. In addition, samples of HKαα/αααanti-4.2, HKαα/αααanti-3.7, and HKαα/HKαα have not yet been collected. So, more samples should be collected and tested to confirm the findings of our study.
Generally, patients carrying the -α3.7 deletion of thalassemia must undergo screening for the αααanti-4.2 and HKαα genotypes because of the high carrier frequency. Using nested PCR combined with MLPA can reduce the misdiagnosis rate of the HKαα allele and -α3.7/αααanti-4.2 and enable more accurate genetic counseling.
This work was supported by a grant from the Department of Science and Technology of Sichuan province, China (No. 30504010332).
Conflicts of interest
1. Weatherall DJ, Clegg JB. Thalassemia
--a global public health problem. Nat Med
1996; 2:847–849. doi: 10.1038/nm0896-847.
2. Lai K, Huang G, Su L, He Y. The prevalence of thalassemia
in mainland China: evidence from epidemiological surveys. Sci Rep
2017; 7:920doi: 10.1038/s41598-017-00967-2.
3. Yu X, Yang LY, Yang HT, Liu CG, Cao DC, Shen W, et al. Molecular epidemiological investigation of thalassemia
in the Chengdu Region, Sichuan Province, Southwest China. Hemoglobin
2015; 39:393–397. doi: 10.3109/03630269.2015.1070733.
4. Wang W, Chan AY, Chan LC, Ma ES, Chong SS. Unusual rearrangement of the alpha-globin gene cluster containing both the -alpha3.7 and alphaalphaalphaanti-4.2 crossover junctions: clinical diagnostic implications and possible mechanisms. Clin Chem
2005; 51:2167–2170. doi: 10.1373/clinchem.2005.054189.
5. Shang X, Li Q, Cai R, Huang J, Wei X, Xu X. Molecular characterization and clinical presentation of HKαα and anti-HKαα alleles in southern Chinese subjects. Clin Genet
2013; 83:472–476. doi: 10.1111/cge.12021.
6. Wang W, Ma ES, Chan AY, Prior J, Erber WN, Chan LC, et al. Single-tube multiplex-PCR screen for anti-3.7 and anti-4.2 alpha-globin gene triplications. Clin Chem
2003; 49:1679–1682. doi: 10.1373/49.10.1679.
7. Abedini SS, Forouzesh Pour F, Karimi K, Ghaderi Z, Farashi S, Tavakoli Koudehi A, et al. Frequency of α-globin gene triplications and coinheritance with β-globin gene mutations in the Iranian population. Hemoglobin
2018; 42:252–256. doi: 10.1080/03630269.2018.1526192.
8. Traeger-Synodinos J, Kanavakis E, Vrettou C, Maragoudaki E, Michael T, Metaxotou-Mavromati A, et al. The triplicated alpha-globin gene locus in beta-thalassaemia heterozygotes: clinical, haematological, biosynthetic and molecular studies. Br J Haematol
1996; 95:467–471. doi: 10.1046/j.1365-2141.1996.d01-1939.x.
9. Mehta PR, Upadhye DS, Sawant PM, Gorivale MS, Nadkarni AH, Shanmukhaiah C, et al. Diverse phenotypes and transfusion requirements due to interaction of β-thalassemias with triplicated α-globin genes. Ann Hematol
2015; 94:1953–1958. doi: 10.1007/s00277-015-2479-8.
10. Steinberg-Shemer O, Ulirsch JC, Noy-Lotan S, Krasnov T, Attias D, Dgany O, et al. Whole-exome sequencing identifies an α-globin cluster triplication resulting in increased clinical severity of β-thalassemia
. Cold Spring Harb Mol Case Stud
2017; 3: doi: 10.1101/mcs.a001941.
11. Farashi S, Bayat N, Faramarzi Garous N, Ashki M, Montajabi Niat M, Vakili S, et al. Interaction of an α-globin gene triplication with β-globin gene mutations in Iranian patients with β-thalassemia
2015; 39:201–206. doi: 10.3109/03630269.2015.1027914.
12. Liu YT, Old JM, Miles K, Fisher CA, Weatherall DJ, Clegg JB. Rapid detection of alpha-thalassaemia deletions and alpha-globin gene triplication by multiplex polymerase chain reactions. Br J Haematol
2000; 108:295–299. doi: 10.1046/j.1365-2141.2000.01870.x.
13. Tan AS, Quah TC, Low PS, Chong SS. A rapid and reliable 7-deletion multiplex polymerase chain reaction assay for alpha-thalassemia
2001; 98:250–251. doi: 10.1182/blood.v98.1.250.
14. Huang Q, Wang X, Tang N, Yan T, Chen P, Li Q. Simultaneous genotyping of α-thalassemia
deletional and nondeletional mutations by real-time PCR-based multicolor melting curve analysis. J Mol Diagn
2017; 19:567–574. doi: 10.1016/j.jmoldx.2017.04.003.
15. Clark BE, Shooter C, Smith F, Brawand D, Thein SL. Next-generation sequencing as a tool for breakpoint analysis in rearrangements of the globin gene clusters. Int J Lab Hematol
2017; 39: (Suppl 1): 111–120. doi: 10.1111/ijlh.12680.
16. Sellner LN, Taylor GR. MLPA and MAPH: new techniques for detection of gene deletions. Hum Mutat
2004; 23:413–419. doi: 10.1002/humu.20035.
17. Taylor CF, Charlton RS, Burn J, Sheridan E, Taylor GR. Genomic deletions in MSH2 or MLH1 are a frequent cause of hereditary non-polyposis colorectal cancer: identification of novel and recurrent deletions by MLPA. Hum Mutat
2003; 22:428–433. doi: 10.1002/humu.10291.
18. Colosimo A, Gatta V, Guida V, Leodori E, Foglietta E, Rinaldi S, et al. Application of MLPA assay to characterize unsolved α-globin gene rearrangements. Blood Cells Mol Dis
2011; 46:139–144. doi: 10.1016/j.bcmd.2010.11.006.
19. Cui J, Azimi M, Baysdorfer C, Vichinsky EP, Hoppe CC. Application of multiplex ligation-dependent probe amplification
to screen for β-globin cluster deletions: detection of two novel deletions in a multi ethnic population. Hemoglobin
2013; 37:241–256. doi: 10.3109/03630269.2013.782461.
20. Nezhat N, Akbari MT. Detection of deletions/duplications in α-globin gene cluster by multiplex ligation-dependent probe amplification
. Genet Test Mol Biomarkers
2012; 16:684–688. doi: 10.1089/gtmb.2011.0251.
21. Suemasu CN, Kimura EM, Oliveira DM, Bezerra MA, Araujo AS, Costa FF, et al. Characterization of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification
in the Brazilian population. Braz J Med Biol Res
2011; 44:16–22. doi: 10.1590/s0100-879x2010007500144.
22. Xie XM, Wu MY, Li DZ. Evidence of selection for the α-globin gene deletions and triplications in a southern Chinese population. Hemoglobin
2015; 39:442–444. doi: 10.3109/03630269.2015.1072551.
23. Xu XM, Zhou YQ, Luo GX, Liao C, Zhou M, Chen PY, et al. The prevalence and spectrum of and thalassaemia in Guangdong Province: implications for the future health burden and population screening. J Clin Pathol
2004; 57:517–522. doi: 10.1136/jcp.2003.014456.
24. Wu MY, Li J, Li SC, Li Y, Li DZ. Frequencies of HKαα and anti-HKαα alleles in Chinese carriers of silent deletional α-thalassemia
2015; 39:407–411. doi: 10.3109/03630269.2015.1071268.