Thalassemia carriers identified by hematological examinations and traditional DNA sequencing
Among 8,962 participants, 1961 subjects were initially identified as thalassemia carriers by hematological examinations, and the detection rate was as much as 21.88%. However, only 988 subjects were confirmed to be thalassemia by traditional DNA sequencing, including 658 α-thalassemia carriers, 294 β-thalassemia carriers, and 36 composite α- and β-thalassemia carriers. The general detection rate is 11.02% (988/8962).
Thalassemia carriers identified by NGS
In contrast, when all 8,962 individuals were directly analyzed by NGS, 1314 subjects were diagnosed as thalassemia carriers, including 875 α-thalassemia carriers, 391 β-thalassemia carriers, and 48 composite α- and β-thalassemia carriers. The overall detection rate of thalassemia is 14.66% (1314/8962). Of them, α-thalassemia carriers and β-thalassemia carriers accounted for 9.76% (875/8962) and 4.36% (391/8962), respectively. In addition, the rate of composite α and β-thalassemia was 0.54% (48/8962), which was determined for the first time in Guizhou (Table 1, Fig. 2).
Alpha- and beta- gene alterations associated with thalassemia
To date, five α-deletion alterations (-α3.7, -α4.2, -- SEA, --FIL, --THAI) and two common large fragment deletions of β thalassemia [SEA-HPFH, Gγ + (Aγδβ)] have been reported. In this study, a total of 17 different α variations with 22 distinct genotypes were identified (Table 1). Of them, --SEA/αα was the most abundant α-thalassemia genotype (26.86%), and -α3.7/αα, -α4.2/αα, and ααCS/αα genotypes are also common and represented 19.66%, 7.77%, and 7.31%, respectively. In addition, rare variations such as αα/-THAI, Poly A (A->G), Hb Phnom Penh, and Initiation codon (-T) were detected for the first time in Mainland, China.
In addition, 16 different β variations with 16 distinct genotypes were detected in this study (Table 1). Of them, βCD41-42/βN and IVS-II-654 (C>T)/βN are the two most common genotypes. This finding is consistent with the previous report [7].The frequencies of the βCD41-42/βN and IVS-II-654 (C>T)/βN were 36.57% and 26.09%, respectively, in the study. The remaining common genotypes include βCD17/βN, β-50/βN, and β-28/βN with corresponding frequencies of 10.74%, 9.72%, and 8.70%. In addition, a rare β-thalassemia mutation β147(HC3)Stop→Gln;HBB: c.442TC) was only found in Guizhou, China and has been named as Hb Zunyi [8].
Among the 48 carriers of composite α and β-thalassemia (22 males, 26 females), 77.08% of genotypes are common deletions of α-globin gene (αα/--SEA, αα/-α3.7, αWSα/αα, αα/-α4.2,) combined with a β-globin gene point mutation. Of the genotypes, composite αα/-α3.7 and Codons 41/42 (-TTCT)/βN was the most frequent one. In addition, 10 abnormal hemoglobin variants including an α-triplication (-αααanti4.2/αα ) combined with the b-globin gene mutation, CD17, were identified in the 48 subjects. Among the 857 subjects of α-thalassemia, 76 subjects were α-triplication carriers (8.87%), including 56 αα/αααanti4.2 and 20 αα/αααanti3.7.
Geographic Distribution of thalassemia gene carriers in Guizhou
Among the 9 regions in Guizhou province, Qiannan was the region with highest carrier rate of α-thalassemia (13.34%, 186/1934), while Guiyang presented the highest carrier rate of β-thalassemia (6.43%, 36/560). Overall, Qiannan presented the highest carrier rate of thalassemia (20.23%, 282/1934), followed by Qiandongnan (17.64%, 339/1922), Qianxinan (15.46%, 107/692), Anshun (14.82%, 126/850), and Tongren (14.49%, 80/552). The difference of the thalassemia gene carrier rates between those regions was statistically significant (P<0.05) (Table 2, Fig. 3).
Ethnic Distribution of thalassemia gene carriers
Among the ethnic groups including Han, Yi, Miao, Buyi, Shui, Tujia, Yao, Gelao, and Zhuang, the ethnic group with the highest carrier rate of α-thalassaemia was Buyi (13.27%, 186/1402), while Gelao was the group with the highest rate of β-thalassaemia (9.09%, 12/132). The highest overall carrier rate of thalassaemia was Zhuang (21.05%, 16/76), followed by Buyi (19.90%, 279/1402), Miao (18.20%, 294/1615), Gelao (17.74%, 185/1043), and Tujia (17.33%, 26/150). The difference of the thalassemia carrier rates between the ethnic groups was also significantly different (P<0.05). (Table 3, Fig. 3).
Identification of high-risk couples with thalassemia by NGS
In this study, we identified 0.85% (38/4881) of the couples with high risk for thalassemia using the NGS, and only 0.36% (16/4481) of the couples was found to be high-risk carriers for thalassemia by routine techniques (Fig. 3, Table 4). Thus, compared with the traditional screening/detection methods, there are 22 (23.2%) more high-risk couples for thalassemia that were identified by NGS. Among the 38 couples, 10 couples carried --SEA/αα genotype and are high-risk for Hb Bart's edema, 11 couples were at high risk for the H disease, and the other 16 couples are carriers of heterozygous mutations. Interestingly, about half of these couples were from the same ethnic group and live in an isolated area of Guizhou Province, suggesting founder variations may exist in this particular subpopulation.
Higher detection rate of thalassemia using NGS than traditional techniques
When traditional techniques including hematological tests and Sanger DNA sequencing were adopted, only 988 subjects were confirmed to be thalassemia carriers, the detection rate was 11.02% (988/8962). In contrast, 1314 subjects were identified to be thalassemia carriers by high-throughput NGS, and the detection rate was 14.46% (1314/8962). There are 326 cases or 3.64% (326/8962) of thalassemia carriers missed by routine detection techniques. Of the 326 cases, 74 were undetectable by conventional thalassemia detection techniques (Table 5), and another 252 were missed due to the lower sensitivity of the hematological examination or the defects of conventional thalassemia gene detection technology. In addition, 38 couples (0.85%, 38/4481) with high risk for thalassemia were identified by NGS while only 16 couples (0.36%, 16/4481) were detected by traditional methods.