| Background and Objectiveβ-thalassemia is one of the most common monogenic diseases worldwide. This inherited hemoglobin disorder is caused by mutations in the humanβ-globin gene (HBB). Up till now, over two hundredβ-thalassemia mutations have been identified in the different populations from endemic regions of this disorder around world. Among them, the vast majority of disease-causing mutations are single base substitutions and insertions or deletions of a few nucleotides, and only a few forms are contributed by partial or complete deletions of theβ-globin gene. In southern China, especially in Guangdong and Guangxi Provinces, there is a high carrier prevalence ofβ-thalassemia, with showing nearly 50β-thalassemia alleles including 43 types of point mutations or small deletions/insertions and 6 types of gross deletions in the southern Chinese.Recently, Huang and colleagues reported a novelβ-globin gene deletion of 1.357 kb in a Taiwanese carrier, who had aβ0-thalassemia phenotype with unusually high levels of Hb A2 and markedly increased Hb F. In this study, we also identified this form of the deletion in five members of a mainland Chinese family by molecular analysis. Interestingly, the chromosome with this deletional form of theβ-thalassemia was confirmed to carry threeγ-globin genes (ε-GY-AGγ-Aγ-ψβ-δ-β-1.357kb). Interaction of this linked mutant gene with theβ+-thalassemia allele -28(A→G) in the affected proband produced a phenotype of (3-thalassemia intermedia (TI). We have assessed correlation between the clinical/hematological phenotype andβ-globin cluster genotype in the individuals with the unusual defects from this family, focused on both the molecular analysis of the defectiveβ-globin gene cluster and the measurement of theβ-mRNA andγ-mRNA levels.Materialss and MethodsSubjectsThe proband, an 11-year-old girl, belongs to a Chinese family that originated from Yunfu county, west of Guangdong Province in southern China. She had a history of fatigue, recurrent dark-colored urine, jaundice and splenomegaly. Her past medical history showed that she was born normally, without exaggerated or prolonged neonatal jaundice and was noticed with an abnormally pale face at 8 months of age. At 6 years old, her spleen was palpated below the left costal and enlarged gradually. She was diagnosed asβ-thalassemia intermedia (TI) at 8 years of age according to the following clinical and hematological phenotypes:she presented with a slight jaundice, short stature, but no frontal bossing or hepatomegaly. Her spleen tip was palpated 4.5 cm below the left costal margin. Laboratory evaluation revealed hypochromic microcytic anemia with a hemoglobin (Hb) level of 9.0 g/dL, a mean cell volume (MCV) of 65.7 fL, a mean cell hemoglobin (MCH) of 22 pg and a red cell distribution width (RDW) of 37.7%. Hb electrophoresis showed a hemoglobin A2 (Hb A2) level of 2.8%, and a hemoglobin fetal (Hb F) level of 72.3%. Hepatic function tests showed that her total serum bilirubin was elevated at 49.9μmol/L (normal range: 6-20), indirect bilirubin 35.6μmol/L (normal range:0-14), direct bilirubin 14.3μmol/L (normal range:0-7).During the subsequent 3 years, her anemia progressed with gradually decreasing levels of Hb from 9.0 g/dL to 5.3 g/dL, but she did not experience any problems in her daily life. She was referred to us for genetic testing at 11 years of age. Upon examination, she showed a proportional short stature, a slight skin yellowing, a mild hepatomegaly, and a severe splenomegaly, but she never received a blood transfusion before visiting us for confirmative diagnosis this time. Obtained the family's consent, we collected peripheral blood samples from 17 family members from three generations for this study.Hematologic MethodsHematological parameters were measured by an automated cell counting and the Hb A, Hb A2 and Hb F levels were measured with an automatic HPLC system. The distribution of HbF in erythrocytes was assessed by an acid elution method. The relative proportion of Gγ, Aγ,βandα-chains were determined by acid-urea polyacrylamide gel electrophoresis.DNA analysisGenomic DNA was extracted from all the peripheral blood samples according to standard laboratory procedures using a phenol/chloroform method. The known Chineseα-thalassemia deletions and point mutations were genotyped using Gap-PCR and the RDB assay, respectively. The 11 knownβ-thalassemia mutations in the Chinese population were identified by the RDB assay. Multiplex ligation-dependent probe amplification (MLPA) was used to detect theβ-globin gene gross deletions. A primer pair that spans the presumed deletion indicated by the MLPA typing pattern was designed to amplify the entireβ-globin gene and its flanking sequence (GenBank Accession NG000007.3) by using Gap-PCR assay. The excision breakpoint of this deletion was further characterized by sequencing of the proper PCR products.In order to define theγ-globin gene rearrangement suggested by the MLPA profiling, we designed a primer pair to amplify the AGγ-hybrid gene that is involved in the triplicatedγ-globin gene, the upper primer is at the position-499 to-475 nt upstream of the Cap site of the Aγ-globin gene, and the lower primer is at the position +2191 to +2210 nt downstream of the Cap site of the Gγ-globin gene. By PCR detection using these two primers, we could not amplify any detectable product in a normal DNA sample, while produced an amplified fragment specific for the AGγ-hybrid gene in the sample with theγ-globin gene triplication. Sequencing of the promoter regions of both the Aγ-globin and the Gγ-globin gene was carried out to identify the nondeletional HPFH orδβ-thalassemia determinants since the variations occurred at these two promoters including the polymorphism of Xmnl site at position-158 in the Gy-globin gene were reportedly associated with elevated levels of Hb F. Haplotype analysis of theβ-globin gene cluster was performed using seven polymorphic sites as described by YJ Lee et al.RNA analysisWe design a real-time quantitative reverse-transcript PCR asaay to evaluate the expression level ofβ-globin gene mRNA andγ-globin gene mRNA in deletion and triplication carriers. On the basis of two standard curves method, we calculate the meanβ-globin gene mRNA andγ-globin gene mRNA relative concentration in four mutation carriers and four normal individuals。The difference of meanβ-globin gene mRNA and y-globin gene mRNA relative concentration beween deletion and triplication carriers groups and normal control group were analysis by the independent samples t test using statistical software SPSS, version 13.0.ResultsHematological data analysisThe proband (â…¢:1) was found to have a TI phenotype with a hypochromic microcytic anemia (Hb 5.3 g/dL, MCV 67.7 fL and MCH 20.9 pg),5.4% Hb A2, 72.8%Hb F and 55.1% Gγ-chain in Hb F, and the distribution of Hb F in her RBC appeared to be pancellular. In addition, her eight other family members (â… :1,â… :3,â…¡:1,â…¡:3,â…¡:5,â…¡:6,â…¡:9 andâ…¢:2) showed hematological features ofβ-thalassemia traits and four of them (â… :3,â…¡:6,â…¡:9 andâ…¢:2) were presenting elevated levels of Hb F range from 4.3% to 11.3% and heterocellular distribution of Hb F, suggesting that the P-globin gross deletion was inherited from her mother.Molecular analysis of the P-globin gene clusterThe results of genotyping of DNA samples in this family were summarized in the last two rightmost columns of Table 1 and Figure 1. The results indicated that the proband was a compound heterozygote with P+-thalassemia -28(A→G) mutation and P-globin gross deletion, in which -28(A→G) was inherited from his father and P-globin gross deletion was inherited from his mother. A total of eight P-thalassemia heterozygotes composed of each half of all carriers with the above two defects were identified in this family. Besides, three family members (â… :4,â…¡:7 andâ…¡:11) were found to carry the silent-α3.7/allele.The MLPA profiling patterns of the proband, her mother and other three matrilineal members (â…¢:1,â…¡:6,â… :3,â…¡:9 andâ…¢:2) showed both the presence of a gross deletion of the P-globin gene and of a triplicatedγ-globin gene arrangement in their genomic DNA samples. Her father and other three matrilineal members (â…¡:5,â…¡:7,â…¡:11 andâ…¢:3) showed the normal signal heights for all probes.Gap-PCR followed by sequencing analysis of abnormal DNA fragments, that span the breakpoint of the presumed deletion revealed a 1.357-kbβ-globin gene deletion starting from-548 bp to +810 bp relative to the Cap site, that is the same as Taiwan deletion reported previously by Huang et al. The PCR detection using AG-FP/AG-RP primer pair confirmed that presence of the AGγ-hybrid gene located between the Gγgene and the Aγgene, generating a triplicatedγ-globin gene arrangement (Gγ-AGγ-Aγ). The detailed sequence analysis of this newγ-globin gene suggested that this AGγ-hybrid gene was generated from fused of the Aγ-globin gene at the 5'region and the Gγ-globin gene at the 3'region. The entire coding region was identical to Gγ-globin gene. It was presumed that the Aγ-Gγhybrid (AGγ-hybrid) was created from a crossover between mismatched chromosomes that produced a y-globin gene triplication with an AGγ-hybrid gene flanked 5'by a Gy-gene and 3'by an Aγ-gene (-Gγ-AGγ-Aγ).The family-based haplotype analysis showed that all five members (â… 3,â…¡:6,â…¡:9,â…¢:1 andâ…¢:2) who carried both a 1.357 kb P-globin gene deletion and a heterozygousγ-globin gene triplication were identified as "+------", thus confirming that the deletion and triplication are on the same chromosome. Therefore, we identified the linkage ofβ-globin gene deletion of 1.357 kb andγ-globin gene triplication (ε-Gγ-AGγ-Aγ-ψβ-δ-β-1.357kb) in this Chinese family. The haplotype linked to the -28(A→G) was "-++-+-+", and a 4-bp deletion (AGCA) at position-225 to -222 nt of the Aγglobin promoter is characteristic of this haplotype, but this small deletion wasn't seen in the AGγ-globin promoter.Real-time quantitative PCR showed the mean relative P-globin mRNA concentration in the 1.357 kb deletion and y-globin gene triplication group were significant lower than in the normal control group (t=4.413, P=0.005). However, the mean relativeγ-mRNA concentration was markedly higher in the 1.357 kb deletion and y-globin gene triplication group than in the normal control group(t=2.281, P=0.031).DiscussionsIn this study, we have performed the molecular analysis of the composite mutant allele that involves aβ-globin gross deletion and aγ-globin gene triplication (-Gγ-AGγ-Aγ) observed in a mainland Chinese family withβ-thalassaemia. Thisβ-globin gene defects was firstly described in a Taiwanese carrier who had a classicalβ-thalassemia trait, showing a 1.357-kbβ-globin gene deletion (denominated a Taiwanese deletion) and a possibleγ-globin gene triplication based on the MLPA analysis. Our present work confirmed their previous findings and firstly identified that the 1.357-kbβ-globin gene deletion and aγ-globin triplication are on the same chromosome by a family-based haplotype analysis. Furthermore, we have characterized theγ-globin gene rearrangement as the -Gγ-AGγ-Aγtriplication using Gap-PCR followed by DNA sequence analysis. Therefore, the P-globin gene cluster bored this composite allele could be recorded as (ε-Gγ-AGγ-Aγ-ψβ-δ-β-1.357kb).The carriers with the composite mutant allele showed the typical haematological phenotypes of aβ-thalassaemia trait except for ones with increased levels of Hb F (range from 4.3% to 11.3%). Comparison of our data with that previously reported for one carrier with this defective gene revealed the similar hematological phenotypes in the heterozygote, who also had hypochromic microcytosis and elevated Hb F and Hb A2 levels (MCV 72.5 fL, MCH 25.2 pg, Hb F 8.9% and Hb A2 6.6%). The blood phenotyping of high levels of Hb A2, a modest elevation of Hb F, and heterocellular distribution of F-cells suggested the exclusion of non-deletional (δβ)0-thalassemia and HPFH for our cases, which is in accordance with the results of no mutations identified in the promoter regions of both the Aγ-globin and the Gγ-globin gene by DNA sequencing analysis.Interaction of this composite mutant allele with theβ+-thalassemia allele -28 (A→G) in the proband gave rise to a TI phenotype for our proband. According to the proband's medical history, she manifested a clinical phenotype of classic thalassemic syndromes and did not require a transfusion as most TI patients during her first 10 years. However, her anemia progressed with gradually decreasing levels of Hb from 9.0 g/dL to 5.3 g/dL and having a severe splenomegaly for the past three years. Her modest clinical phenotypes should be attributed to her unique causative genotype, which involved genetic factors both ofβ+-thalassemia allele for expressing low levels ofβ-globin chains and of this linked mutant allele for some compensatory Hb F. She has shown severe clinical symptoms and required regular blood transfusion since she was 11 years of age. Her severe clinical condition might be associated with a severe splenomegaly with hypersplenism.The markedly high levels of Hb F in the four (-Gγ-AGγ-Aγ)-β-1.357kb-thalassemia heterozygotes should mainly be attributed to the increased expression ofγ-globin genes caused by P-globin gene gross deletion including the promoter region, which altered chromatin structure and competition with the remaining y-globin gene promoters for limited transacting factors and the LCR. Theγ-globin gene competes more favorably for interaction with the LCR in the absence of theβ-gene promoter. In addition, theγ-globin gene triplication (-Gγ-AGγ-Aγ) linked to theβ-globin gross deletion might contribute to the elevated level of Hb F in the child (proband) although previous available studies showed that adults with a singleγ-globin triplication had normal levels of Hb F and low Gy-chain values. The affected proband had a higher y-chain value (55.1%) than that of the four heterozygotes, suggesting that the additional AGy-hybrid gene could produce Gγ-chains to some degree; this phenomenon revealed that the greatly increased production of theγ-chain could depend on the anemic condition of the subject. Moreover, the role of the XmnI sites at position -158 of the Gγ-globin gene could be ruled out according to our observation.This is the firstly identified report of a P-globin gross deletion linked to a y-globin gene triplication; their genetic linkage would result from the two independent events (non-homologous recombination and unequal crossing over) on the same chromosome but at different times. No sequence homology was found when the junction fragment was aligned with the normal sequences both at the 5'end and at the 3'end of around the breakpoints. However, an AT-rich region which could be related to DNA breakage during non-homologous recombination events, was found around the 5'breakpoint of the deletion; therefore, a non-homologous recombinant event might be responsible for the deletion. The -Gγ-AGγ-Aγtriplication could have arisen from an unequal crossing over between the position -271 of the Aγ-globin gene and the codon 136 of the Gγ-globin gene. However, it was difficult to determine the accurate 5'and 3'endpoints of crossing over because the sequences of the Gγ-and Aγ-genes are highly homologous. The further study on the mechanism of the linkage ofβ-gene deletion andγ-triplication presented from our work would be valuable to elucidate the regulation of humanγ-globin gene.
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