Guangzhou is located in the center of southern China and consists of eleven districts. Although the population composition of Guangzhou has changed constantly due to frequent migration from other provinces to this place, people with local residential registration were the largest group in the investigated population, ensuring good representation. This report is the first of its kind to describe the prevalence of (δβ)0-thal/HPFH in Guangzhou. Chinese Gγ (Aγδβ)0 thalassemia and SEA-HPFH are the most common deletional (δβ)0-thal/HPFH disorders in this region. The prevalence of these two disorders was lower than that in the Chinese Zhuang population but higher than that in the Chinese Hakka and Yunnan populations [9,10,11]. These differences may be caused by the population composition, which is rather homogeneous in those regions. Guangzhou natives primarily include Cantonese and Hakka population, which live scattered in this city, except for the Huadu, Zengchen and Conghua districts. Approximately 30% of the Guangzhou population came from other parts of the province and outside the province. The top five provinces from which immigrants moved were Guangxi, Hunan, Jiangxi, Sichuan, and Hubei. Deletional (δβ)0-thal/HPFH has seldom been reported in provinces other than Guangxi, which will have an effect on the distribution of deletional (δβ)0-thal/HPFH in Guangzhou. Different distributions of deletional (δβ)0-thal/HPFH depend on the composition of the population in 11 districts, especially in the outer suburb and the newly-established district. As a newly-established district, Nansa district provides many preferential policies and facilitation measures to attract people to settle down in this area. The population composition are mainly immigrants.
However, other outer suburbs, such as Huadu district, Guangzhou natives are the main population in these suburbs. In addition, there is a large number of ethnic minorities, including Zhuang and Tujia, in these outer suburbs. Huadu is also the district in which most Hakka people reside. Further research about the effect of ethnic diversity on the carrier rate of these disorders should be conducted by collecting detailed demographic information.
Chinese Gγ (Aγδβ)0-thal, SEA-HPFH, the Taiwanese deletion, and the Aγ-196C-T mutation are common genetic factors related to high HbF levels in Guangzhou. Only individuals with the Aγ-196C-Tmutation had normal values of MCV and MCH. The Taiwanese deletion and Chinese Gγ (Aγδβ)0-thal manifested the phenotype of beta0-thalassemia, and the values of Hb, MCH and MCV were much lower than normal range. The HbA2 level was lower than 3.5% in heterozygous carriers of Chinese Gγ (Aγδβ)0 thal or the Aγ-196 C-T mutation, whereas the latter carriers have considerably lower HbA2 levels, regardless of coinheritance with α0-thalassaemia (Southeast Asian type deletion(−-SEA/αα)) heterozygosity. The mechanism regulating this phenomenon is not clear because the Aγ-196C-T mutation only involves a point mutation in HBG1. This mutation is difficult to discriminate among carriers with Southeast Asian type deletion (−-SEA/αα) coexistence due to similar hematological indicators. The HbA2 level was higher than 3.5% for cases of heterozygous SEA-HPFH mutation or Taiwanese deletion, and individuals with SEA-HPFH deletion had normal, hypochromic or borderline red blood cell indices. To date, the Taiwanese deletion has only been reported as the case study, [18, 19]. Compared to the three types, that is, Chinese Gγ (Aγδβ)0-thal, SEA-HPFH andAγ-196 C-T mutation [9, 10, 20], the individual with the Taiwanese deletion showed higher HbA2 levels than 6% of our study cohort. This higher HbA2 level may due to deletion of the β-globin gene promoter. A competitive relationship exists among the β, γ and δ chains in the HS region. Aγ-globin gene triplication was observed among our cases, as has been described before. All four of these cases exhibited a high HbF level of more than 8%; in contrast, the HbF level in the case without γ-globin gene triplication was only 3.4%, which was lower than that of the other cases. Whether γ-globin gene triplication contributes to the elevated level of Hb F in the presence of the Taiwan deletion should be further studied. It has been previously described that compound Taiwan deletion with a mutant allele with β-thal generates the β-TI phenotype. Thus, it is necessary to identify Taiwanese deletion carriers in those areas with a high prevalence of thal. The Taiwanese deletion may constitute the best possibility of when no β-globin gene mutation is found in a case of increased HbF and Hb A2 (> 6%).
Only one type of Hb Lepore variant, known as Hb Lepore-Boston/Washington, has been reported in China . Fusion between the HBD gene and the HBB gene occurs due to their high sequence homology. Homozygotes for Hb Lepore or compound heterozygotes for Hb Lepore and β-thalassemia can present with thalassemia major or intermedia. Hb Lepore is highly important for diagnosis, with levels ranging from 5 to 15%.Hb Lepore-Boston/Washington has been described in Thailand . Compared to HPLC (High Performance Liquid Chromatography), the capillary electrophoresis system is a better method for identifying cases with Hb Lepore and Hb E by a peak of denatured Hb E in zone 6, therefore, we can discern compound Hb Lepore/Hb Efrom Hb E/Hb E. It was reported that carriers with Hb Lepore-Boston/Washington showed slightly elevated Hb F levels, which was not consistent with our observations. More cases are needed to illustrate the molecular and hematological characteristics of this disease.
We found a case with the β− 28/β Chinese Gγ (Aγδβ)0 genotype, a high level of HbF and mild anemia. So CC  described a patient with the same genotype who presented moderate anemia. The phenotype of patients with the β+/βChinese Gγ(Aγδβ)0 genotype or β0/βChinese Gγ(Aγδβ)0 genotype varies, making genetic counseling challenging. There is also some debate regarding pregnancy termination in the case of a fetus with those genotypes.
Three cases of β-globin gene cluster deletions were found by MLPA. Case 1 exhibiting the HBG2-HBG1 fusion gene, was similar to a case reported by Seung-Tae Lee, but the breakpoint may be different . The fusion occurred between HBG2 exon 2 and HBG1 IVS2 in our study, but it consisted of a HBG2-derived 5′ part and a HBG1-derived 3’part in Seung-Tae Lee’s paper. A positive regulatory region,Aγ-IVS2, has been reported before, and it might influence HbF regulation in adults . The HBG2–HBG1 fusion can cause γ-thalassemia, which has been described before . The breakpoints displayed heterogeneity because the HBG2 and HBG1 genes are highly homologous. The mechanism of γ-thalassemia caused by HBG2–HBG1 gene fusion should be further explored. The functional study of HBG2–HBG1 gene fusion may be performed in K562 cells and HUDEP-2 cells exhibiting embryonic/fetal and adult patterns respectively.
Nondeletional-HPFH has not been described systematically in China. Italian nd-HPFH (Aγ-196 C-T mutation) was first described in 1984 , but it has seldom been reported ever since that. Our study found that Italian nd-HPFH was the most common nondeletional HPFH in Guangzhou, of which the familial prevalence was 0.042%. Because Italian nd-HPFH and Chinese Gγ (Aγδβ)0-thal carriers have similar HbF and HbA2 levels, the red blood cell count is the only difference used for discrimination during initial screening for thalassemia. We first detected one Italian nd-HPFH heterozygous condition coexisting α-thal and β-thal, with high HbA2 and HbF levels being detected (HbA2:4.1%, HbF: 25.9%), which is similar to SEA-HPFH carriers. Overall, the phenotypic characteristics of Italian nd-HPFH are useful for genetic counseling for individuals with increased HbF. We also found another nd-HPFH, known as Cretan HPFH (Aγ-197 C-T), which was described in 2014 . The two types of nd-HPFH are related to point mutations at approximately − 200 relative to the transcriptional start site of the fetal γ-globin gene. Using K562 cells and HUDEP-2 cells, Martyn et al. revealed that mutations at − 200 of the γ-globin promotor disrupt ZBTB7A for HbF repressor binding . These researchers introduced c.–195C > G into HUDEP-2(Δ Gγ) WT cells by CRISPR–Cas9 and found that fetal hemoglobin protein levels increased to approximately 24.0%. The results indicated that homozygosity of engineered HPFH cells could cause higher HbF levels than patients with heterozygous HPFH-associated mutations. It was indicated that introducing naturally occurring variants in this region may be considered as an attractive gene therapy strategy. Coincidentally, we detected two homozygotes for the Aγ-196 C-T mutation that, to the best of our knowledge, had not been reported previously, raising the possibility that the HbF levels of homozygotes can be similar to those of heterozygotes. Our study is the first to describe a patient in which the binding of ZBTB7A is disrupted on both alleles. There may be some causes for these differences: ZBTB7A may work with additional cofactors at different stages of cell differentiation; the degree to which ZBTB7A influences HbF levels is related to erythropoietic stress, which is characteristic of β-globin chain deficiency. More research is needed to estimate the impact of homozygous and heterozygous Aγ-196 C-T mutations on HbF levels, especially when coexisting with major β-thal. If the effect is similar, we can achieve the expected treatment result simply by introducing heterozygous HPFH-associated mutations.