Molecular epidemiology and hematologic characterization of δβ-thalassemia and hereditary persistence of fetal hemoglobin in 125,661 families of greater Guangzhou area, the metropolis of southern China

Abstract

Background: Individuals with δβ-thalassemia/HPFH and β-thalassemia usually present with intermedia or thalassemia major. No large-scale survey on HPFH/δβ-thalassemia in southern China has been reported to date. The purpose of this study was to examine the molecular epidemiology and hematologic characteristics of these disorders in Guangzhou, the largest city in Southern China, to offer advice for thalassemia screening programs and genetic counseling.

Methods: A total of 125,661 couples participated in pregestational thalassemia screening. 654 subjects with fetal hemoglobin (HbF) level ≥ 5% were selected for further investigation. Gap-PCR combined with Multiplex ligation dependent probe amplification (MLPA) was used to screen for β-globin gene cluster deletions. Gene sequencing for the promoter region of HBG1 /HBG2 gene was performed for all those subjects.

Results: A total of 654 individuals had hemoglobin (HbF) levels≥5, and 0.12% of the couples were found to be heterozygous for HPFH/δβ-thalassemia, including Chinese ^Gγ (^Aγδβ)⁰-thal, Southeast Asia HPFH (SEA-HPFH), Taiwanese deletion and Hb Lepore-Boston-Washington. The highest prevalence was observed in the Huadu district and the lowest in the Nansha district. Three cases were identified as carrying β-globin gene cluster deletions, which had not been previously reported. Two at-risk couples (0.0015%) were required to receive prenatal diagnosis. We also found 55cases of nondeletional-HPFH (nd-HPFH), including 54 with Italian nd-HPFH and one with the ^Aγ-197C-T heterozygous state. It is difficult to discriminate between Chinese ^Gγ (^Aγδβ)⁰-thal and Italian nd-HPFH carriers using hemoglobin (Hb) analysis.

Conclusions: This study is the first to describe the familial prevalence of HPFH/δβ-thalassemia and the high-risk rate in Greater Guangzhou Area, and the findings will support the implementation of thalassemia screening for three common deletions by gap-PCR. We also presented a systematic description of genotype-phenotype relationships which will be useful for genetic counseling and prenatal diagnostic services for β-thalassemia intermedia.

Keywords: Guangzhou; HPFH; Prevalence; δβ-thalassemia.

Fig. 1

Spectrum of δβ-thalassemia and Hereditary Persistence of Fetal Hemoglobin in 654 subjects with HbF>5%

Fig. 2

Structures of the common deletions and the locus of the (^Aγ-196 C-T) mutation identified, LCR, locus control region

Fig. 3

Map showing the prevalence of Chinese ^Gγ (^Aγδβ)⁰ thalassemia and SEA-HPFH in Eleven districts of Guangzhou. (The number and percentage of those two types of δβ thalassemia/HPFH are indicated in detail). The carrier rate of these two disorders in eleven districts of Guangzhou is shown in Fig. 3. It was 0.06% in Guangzhou, which was similar in most districts except for Huadu and Nansha.

Fig. 4

Hematological parameter analysis of heterozygotes of Chinese ^Gγ (^Aγδβ)⁰ thalassemia, SEA-HPFH, Taiwanese deletion, and Italian nd-HPFH. Comparisons of hemoglobin (Hb) (a), mean corpuscular volume (MCV) (b), mean corpuscular hemoglobin (c), hemoglobin A2 (HbA2) (d), and fetal hemoglobin (HbF) (e) levels of those four types of δβ-thalassemia/HPFH with or without coinherited α⁰-thalassaemia (southeast Asian type deletion). Mean ± standard deviation (SD) isused to describe hematological parameters. A p-value of less than 0.05 showing significant differences: **p < 0.01; *p < 0.05

Fig. 5

Chromatograms of point mutations in the proximal fetal γ-globin gene promoters detected in individuals. (a) Cretan HPFH (^Aγ-197 C-T); (b) Homozygote for (^Aγ-196 C-T) mutation

Fig.6

(a) PCR primers around probes with normal dosages in the HBG1- IVS2 and HBG2 upstream regions resulting in the amplification of an abnormally shortened PCR product (1.7 kb) in the patient (lane Patient); (b) MLPA analysis showing half dosages for three probes located in the HBG2 and HBG1 regions in the patient; (c) complete sequences of the HBG2–HBG1 fusion gene in Patient 1. The HBG2 and HBG1 region were composed of two parts: one included the promoter, exon 1, intron 1 and a part of exon 2 of HBG2, and the other included a part of exon 2 and intron 2 of HBG1; HBG2-specific sequences are highlighted in yellow, and HBG1-specific sequences are highlighted in red

Fig. 7

(a) MLPA analysis showing half dosages for two probes located in HBG1 in patient 2 and fourteen probes located in the HBB and HBD region in patient 2; (b) MLPA analysis showing half dosages for two probes located in HBG1 in patient 3