| IntroductionHemoglobin is the transport body of oxygen, which composed by hems and globins. All the normal hemoglobines are tetramers of two pairs of unalike goblin chains these are two a-globin chains (αξ) and two non-α-globin chains (βγδε). There are several a-like and β-like globin genes that are developmentally in different developmental stages at different times regulated to produce embryonic haemoglobins (ζ2e2, HbGowerl, ζ2y2, HbPortland, a2s2 HbGower2) and foetal (a2Gy2, a2Ay2) and adult haemoglobins (α2β2 HbA, a282 HbA2). The y chain has two subtypes, named Gy and Ay, thus, there are two types of HbF:a2Gy2 and a2Ay2.These two are highly similar, except that the 136 acid of the former is glycine, compared with the latter of alanine.Normal adult red cells contain 96.5～97.5% HbA, with 2.5～3.5% of the minor component HbA2 and a small amount (<1%) of HbF. The levels of Hb F accounted for 90% of circulating hemoglobin at birth usually decline a few months after birth, which decreased synthesis begins its third trimester. While in the first year of life, Hb F is gradually being replaced by HbA. The amount of HbF is more than 2% that is called raised HbF levels in normal adult, and hereditary persistence of fetal hemoglobin (HPFH) is characterized by persistent high levels of HbF in adults.The proportion of the two types of y chains of Hb F (Cγ and Aγ) is resulting in a Gy:Ay ratio similar to that seen at birth (70:30), and at a later age which similar to that seen at birth (40:60).The molecular basis of raised HbF levels are some factors that can affect these three (α, β, and y) globin chains, which can lead to imbalance of composition of the threes. An important doctrine of raised HbF levels is the inherited disorders of β like globin gene expression, such as β thalassemia. In adults, the β-globin gene is principal and nearly 97% of all hemoglobin is comprised Hb A. Thus, y-globin genes express poorly and less than 1% Hb F makes up of adult hemoglobin. As disorders of like β-globin gene expression, y-globin genes are highly expressed that leads to excessive amounts of y chains synthesized. The point mutations of y globin gene can produce an enhancer element GGGPyG (Py=pyrimidine base), which can promote binding capacity of generating, or has similar features with the CACCC box, as the capacity of binding protein factors CPI was enhanced, or else, the mutation can reduce function for CAAT box which may act as a negative of the effectiveness of the promoter, so that can increase the expression levels of y genes, as enhance the efficiency of y-globin. The a-thalassemia is as well known a gene disorder, there exists a relationship between clinical phenotypes and genotypes, involving the copy number changes in the human a-globin gene cluster. A normal adult has four copes of a-globin gene due to existing two copies of chromosome 16. Copy numbers in the human a-globin gene cluster increase will produce five copies (aaaanti3./aa or aaaanti4.2/aa), and this additional a-globin gene can produce excessive a-chain, which can aggravate imbalance for a and p-chain, so that will increase clinical symptoms in patients with P-thalassemia. Genetic modifications in raised HbF level play an important role. Gγ-polymorphism (such as position-158 in the Gy-globin gene) and quantitative trait loci (QTL) mapping elsewhere in the genome (such as Xp 22.2-22.3,6q23 and 8q) with association studies recently may play a role in the raised HbF levels, which suggest that a variant between the HBS1L and c-MYB genes is implicated in the QTL for HbF at 6q23. Associated with HbF quantitative traits involved in regulation of y-globin gene expression is closely associated with increased HbF.Currently, foreign researches for increased HbF levels develop in good state orderly, and most reports for factors of increased HbF levels found elevated levels of HbF among different ethnic groups, where there are differences between the genetic modifications. The researches for increased HbF levels of the domestic mechanism are still at infancy in our country, fewer systematic researches, and more studies of Study of a single factor.The objects of this research:(1) To elucidate the molecular mechanism of increased HbF levels systematically and enrich mechanism of cases of increased HbF levels in the Chinese population.(2) To analyze the defection of increased HbF levels with gene disorders in β-like globin genes cluster for cases with high of HbF levels in depth, specifically mutations in the β gene cluster provided us with a more sophisticated understanding of the molecular basis for the persistent y-gene expression.(3) To detect copy number changes of a-globin gene, and analyze molecular mechanisms of the impact of HbF levels for a-globin gene copy numbers loosed or increased.(4) To analyze the (C-T) variation at position-158 upstream of the Gy globin gene that is detectable by XmnI. The role of increased Hb F levels of this sequence variation will be analyzed in the selected cases.(5) For correlation analysis between gene mutations and increased HbF level.Materials and MethodsA total of 85 cases attending the diagnosis for anemia were recruited in Nan Fang hospital, Southern medical university. All of them have increased HBF levels, which were greater than 5%, and their ages were older than two years, no suffering under regenerative anemia and not including pregnant women.RDB was used to detect common Chinese people 17 point mutations in β- globin gene cluster, and the largest deletions of the P-globin gene cluster was detected by MLPA. SEA -HPFH and Chinese δβ thalassemia were validated by Gap-PCR and Sanger sequencing to analyze the full length of the β-globin gene sequencing. The mutations of a-globin gene cluster were analyzed by using the same experimental method. To analyze the Gy-158 (C-T) variation that is detectable by XmnI. Correlation analysis was used to analyze differences between gene mutations and increased HbF level.ResultsAmong 85 cases with raised levels of Hb F levels,12 cases with large deletions of the β-globin gene cluster were identified, of which,9 cases were Chinese Gy (Ayδβ)0,2 cases were SEA-HPFH, and lcase was Ducth I.There were 22 cases identified with heterozygous mutation and 34 cases of compound heterozygous, while,6 cases with homozygous mutation, which were detectable by the variety of experimental methods in the 85 selected samples.This research also found 3 cases of compound heterozygosity for severe β0 or β+ and large deletions in β gene cluster, and 2 cases of homozygous β+thalassemia with and SEA deletions of a gene, lcases of compound Heterozygous for pIVS-2-654 and small deletion in α-gene cluster and lcases of heterozygous β-thalassemia for CD41-42 with aaaant3.7.1 sample of deletions and 1 case of repeat ina -gene cluster were detected.ConclusionDue to the gene disorders(including point mutation and large deletions) in β-globin cluster, the gene expressions of both δ and β genes are at low level, and y-globin genes are highly expressed that leads to excessive amounts of y chains synthesized, that leads to increased HbF levels.Copy numbers in the human a-globin gene cluster increase will produce five copies, so that additional a-globin gene can produce excessive a-chain, which can aggravate imbalance for a and P-chain, so that will increase clinical symptoms in patients with β-thalassemia. Yet, copy number loss or mutation ina-globin gene will cause a corresponding clinical phenotype.In normal healthy, the presence of the polymorphism G y-158 (C>T) can lead to low levels of HbF. Moreover, HbF can also be variably increased in association with β-thalassemia.The large deletions in β-gene cluster have higher HbF levels than heterozygous mutations in β-gene, and there are not significant differences of raised HbF levels between compound heterozygosity for severe β0 or β+ and Homozygous β0 or β+.The effects of increased HbF levels of some SNPs detected by sequencing are still not clear. The inadequacies of this research are that the QTL and the promoter of Gy and Ay gene which are closely associated with increased HbF are not analyzed. |
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