Identification Of A Novel β Thalassemia Mutation And Development Of A Method For High-throughput Mutation Detection Of β-globin Gene

The β thalassemia is one of the most common single gene disorders in the world, with high gene frequency in many areas of the world as a result of malarial selection that is now generally accepted. Therefore, the main distribution regions of β thalassemia are the tropics and subtropics with high incidence of malarial, extending from the Mediterranean basin and parts of Africa, through the Middle East, the Indian subcontinent, South China, South-east Asia and Melanesia and out into the Pacific. In addition, the USA and some countries in Central and South America that had substantial immigration from Europe and Africa, also have a high incidence of β thalassemia. It is well known that the severe forms of β thalassemia major is a fatal disorder, and the patients need regular blood transfusion and desferal therapy to sustain their life. Recently, the thalassemia still poses serious public health issues in these areas. As for a country or a region with high prevalence of thalassemias, the most effective method to control this disorder is to establish a prevention program based on the genetic epidemiology study, which is to implement a large-scale population screening and prenatal diagnosis of high-risk couples to prevent the birth of infants with severe thalassemia.In China, the provinces located in the south of the Yangtze River have a relatively high incidence of β thalassemia, especially Guangdong, Guangxi, Hainan, Jiangxi, Taiwan and Hong Kong with a total population over two hundred million people. This study is trying to find out novel β thalassemia gene and elucidate its genetic basis in the molecular epidemiology investigation and clinical practice of β thalassemia. In addition, the other part of this study is to develop a method for high-throughput and automatic detection of Chinese β thalassemia common mutations by using the latest technology platform in the world. Our results could provide valuable genetic data and detection method for genetic counseling, prenatal diagnosis and carrying out large-scale population prevention program of β thalassemia.1. Identification of a novel P° thalassemia geneThe P thalassemias are a heterogeneous group of the inherited hemolytic anemia mainly resulting from single base substitutions, minor deletions or insertions within the P-globin gene in the chromosome 11, and are classified into (3+ and (3° varieties, reflecting a reduced or an absent synthesis of the P-globin chain of hemoglobin, respectively. More than 200 different mutations of P thalassemia to date have been reported in the world, and each ethnic population has its own cluster of common mutations. In Chinese population, P thalassemia is a common disorder with 33 types of mutations up to date. These mutations can be subdivided into three types according to their functionanl effect, i.e. mutations affecting P-globin gene transcription, mutations affecting mRNA processing and mutations affecting mRNA translation, respectively. The latter include initiation codon mutation, nonsense mutation and frameshift mutation. Frameshift mutations of P-globin gene generate quite frequently a new premature termination codon (PTC) downstream and lead to the synthesis of a truncated PTC protein that has lost its function. The phenotype resulted from PTC mutation is generally the P°-thalassaemia because there is no normal synthesized functional protein. The common mutations, such as CDs 41-42 (-TCTT), CDs 71/72 (+A), CDs 27/28 (+A) and the rare mutation, CDs 14/15 (+G), all can result in P°-thalassaemia. In this study, a novel frameshift mutation in P-globin gene, CDs 15/16, +G; TGG GGC—>TGG G GGC, was discovered in a Chinese family originated from Guangzhou by phenotype analysis of hematological data and direct DNA sequencing.The proband (a female infant aged 2 years 8 months) was diagnosed as transfusion dependent p thalassemia major. According to the results of complete blood count and hemoglobin electrophoresis analysis, we confirmed that the proband's father, mother and maternal grandfather have a trait of microcytic hypochromic, i.e. with reduced mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) (that are, 68.9fL/22.1pg, 56.4fL/19.0pg, 59.1fL/19.9pg, respectively) and an elevated level of HbAa (all >3.5%). The three subjects were characterized as heterozygous P thalassemia (i.e. P thalassemia carriers). With the support of phenotype data, we performed the DNA test by using reverse dot blot (RDB) method to detect the 24 types of Chinese P thalassemia mutations. The result of RDB test showed that the proband and her father carried the transcription mutation of -28(A—>G). However, negative data was presented in testing the DNA of the proband's mother. Therefore, the mutation on the proband's second allele could not be determined. This result indicated that there may be an unknown mutation. Consequently, the mutation analysis of P-globin gene was performed by direct DNA sequencing of the amplifiedPCR product. DNA sequencing analysis of the proband and her mother and maternal grandfather identified a mutation, a single nucleotide insertion of guanine between the codons 15 and 16 within a mutation hot spot region of the P-globin gene, which resulted in a frameshift and a premature terminator at codon 22. Sequencing of the antisence strands did confirm the insertion mutation. By searching the literatures in PubMed and the online database of human haemoglobin variants and thalassaemias in the website of the Globin Gene Server (http://globin.cse.psu.edu/), the PTC mutation of CDsl5/16(+G) was confirmed to be a novel p thalassemia mutation, which was unreported previously. In summary, the proband is a compound heterozygous p thalassemia patient. The causative mutations on her two P thalassemia chromosomes are: the transcription mutation of -28(A—*G), which is inherited from her father and the frameshift mutation of CDsl5/16(+G), which is inherited from her mother and maternal grandfather, respectively. It is obvious that the genetic pattern of P thalassemia gene of this family belongs to classical autosomal recessive inheritance.By analyzing the sequences around the novel mutation of CDsl5/16(+G), we found an interesting event that a homonucleotide runs of four Gs within a highly GC rich region was changed into five Gs due to this 1-base insertion mutation. It is well known that the simple tandem repeat sequences are a hot spot of frameshift mutation. The unstable constitution of tandem repeat sequence is resulted from the event of "slip" in the DNA replication while the DNA polymerase across the repeat sequence, some homonucleotide runs, such as 5Gs or lOCs, et al. If a slippage event is not repaired by the postreplicative mismatch repair (MMR) mechanism before the next round of DNA replication, it will give rise to a deletion or to an insertion. The frameshift mutation of single base insertion of "G" occurs most frequently in tracts of a tandemly repeated nucleotide. Therefore, we can conclude that the most likely mechanism resulted in this CDsl5/16(+G) mutation is DNA replication slippage according to these knowledge and recent study.Frameshift mutation generally results in an unstable mRNA and make its level reduced. A nonsense-mediated mRNA decay (NMD) pathway probably plays an important role in degrading the nonsense transcripts. NMD is a surveillance mechanism by which cells recognize and degrade mRNAs containing the PTC generated by frameshift mutation. In order to evaluate the functional effect of this novel PTC mutation, real-time quantitative RT-PCR assay and relative standard curve method were used to analyze the relative levels of P-globin mRNA of the novel mutation carriers and a group of p+ thalassemia carriers compared with the normal individuals.By double dilution method, the generated linear regression equations describing therelative standard curves were y=-3.4924x+21.6465 (R2=0.998) for a-mRNA and y=-8.2483;c+28.5954 (R2=0.999) for P-mRNA, respectively. After normalization with these equations, the calculated mean Ct values of P-globin mRNA were 14.65±0.13 (n=2) for heterozygous subjects carrying CDsl5/16 (+G) mutation, 13.93±0.28 (n=5) for patients with P+-thalassemia trait and 12.83±0.48 (n=8) for normal individuals, respectively. The results of statistical analyses by using the SPSS 10.0 software package demonstrated a significant differences (P<0.05) between the three groups by pairwise multiple comparison or as a whole. Then, the relative P-globin mRNA levels of two patient groups compared with that of normal control group can be figured out by using the mean Ct values. These results showed significantly decreased amount of P-globin mRNA in patient groups, i.e. the P-mRNA concentration of proband's mother and grandfather had a 39.83% reduction and the 5 p+-thalassaemia heterozygotes had a 26.44% reduction compared with that of normal individuals. These data indicated that the reduced level of P-mRNA of CDs 15/16 (+G) mutation carriers was larger than that of heterozygotes. Therefore, the thalassaemic phenotype resulted from the PTC mutation of CDsl5/16(+G) should be p°.Haplotypes of the P-globin gene cluster is an important genetic background data of P thalassemia gene. We performed the haplotype analysis of 7 classical polymorphic restriction enzyme sites by using PCR-based RFLP (restriction fragment length polymorphism) method and family linkage study. The RFLP haplotypes of the proband's two P thalassemia chromosomes were determined. The haplotypes linkedto the CDsl5/16(+G) mutation and -28(A-?G) were "+---------+ -" and '- + + - + -+", repectively. The former bears a certain frequency both in the P chromosome and in the pA chromosome. The latter is the same haplotype seen in 9 P thalassemia chromosomes contained the -28(A—?G) mutation reported by another study group.This demonstrates that the "- + H-----1- - +" haplotype is highly associated with the-28(A—>G) mutation.In addition, we developed the reverse dot blots method for genotyping the novel mutation of CDsl5/16(+G) by adding a pair of preferentially selected probe and the results indicated high specificity. The integration of this new mutation site extended the detection range of RDB assay, which would be applied conveniently in the molecular diagnosis of P thalassaemia, especially in the prenatal diagnosis required by the at-risk couple (proband's parent) in their nearly future pregnancies.2. The development of a method for high-throughput mutation detection of P-globin gene based on fluorescence polarizationJust as we know, P thalassemia major is a fatal inherited disorder, which requiresregular blood transfusions and lifelong medical care. At present, the most effective method to prevent and control this disorder is large-scale population genetic screening and prenatal diagnosis of couples at high risk. Therefore, an accurate, rapid, high-throughput and automatic mutation detection method is needed for P thalassemia diagnosis in clinical practice.Up to date, there are 34 types of P thalassemia mutations found in Chinese population. But a few common mutations account for the majority of P thalassemia causing alleles. In this study, we developed a homogenous, fast, accurate and high-throughput method for simultaneous detection of the 8 most common mutations (CDs 41-42 (-TCTT), IVS-2-654 (C-VT), -28 (A-?G), CD 17 (A->T), CD 71/72 (+A), CD26 (G-+A), -29 (A-Kj) and CD43 (G-?T), account for -95% of the constitution) of Chinese p thalassemia. This method is a template-directed single base primer extension (SBE) assay coupled with homogeneous fluorescent polarization (FP) detection. The basic principle and experimental steps of our SBE-FP method are stated below.Firstly, 3 pairs of high-specific PCR amplification primers and 8 SBE primers were designed according to the sequences of p-globin gene and the 8 mutation sites. The SBE primers were designed to have a 3' end immediately 5' upstream of the mutation site. For simultaneous detection, SBE primers were also designed into having nearly same melting temperature (Tm) close to 66°C. There were four steps of our SBE-FP method. At the first step, three P-globin gene fragments containing all of the 8 mutation sites were obtained under the same condition by using a little volume (5ul) and high-efficiency (Gold Taq polymerase) PCR amplification system in a black 384-well PCR plate. Next, an enzymatic cocktail of 2[xl containing shrimp alkaline phosphatase and Escherichia coli exonuclease I was added to the PCR product to degrade the residual primer and dNTP. The mixture was incubated at 37°C for 30 min before the enzymes were heat inactivated at 80°C for 15 min. Then, a 13 ul of SBE reaction mixture was added to the 7 ul of enzymatically digested PCR product and the second round of PCR was performed immediately. The SBE mixture contained 5 pmol allele-specific SBE primer and two allele-specific dye-labeled dideoxyribonucleoside triphosphates (for instance, RllO-ddGTP and TAMRA-ddATP for a G/A mutation) and a commercially available modified DNA polymerase. The dye-labeled ddNTP specific for the allele extended the SBE primer (20-30nt) by only one base, with a ~ 10-fold increase of the molecular weight of the fluorophore. Because the fluorophore linked to a large molecule has slower motion (rotates and tumbles), the emission maintains to be polarized (high FP value, FP is proportional to the molecular weight) after excitated by a polarized excitation light. On the contrary,because the dissociative ddNTP (the other dye labeled) remains faster motion, the emission is depolarized (low FP value). Finally, the SBE reaction mixture was directly measured by FP counter without separation or purification. We could determine the genotype of a mutation according to the FP values of the two fluorophores. The samples have high FP for RllO-ddG but low FP for TAMRA-ddA, which indicats that they are of homozygous G genotype. The heterozygous G/A samples exhibit high FP values in both RllO-ddG and TAMRA-ddA. The samples with low RllO-ddG but high TAMRA-ddA values are homozygous A/A.In order to optimize reaction conditions, the kinetic experiments were performed aimed at the key parameters in two rounds of PCR of the SBE-FP assay. The main results were as follows: (1) In the first round of PCR, the quantity of temple (genomic DNA), 4-8 ng/5ui; primers, 100 nM; (2) In the second round of PCR, an anneal temperature of 65°C for all 8 SBE primers and 11-16 PCR cycles for SBE reaction. In addition, a total of 100 random clinical samples previously characterized by RDB test were used to evaluate the specificity of this method by blind experiment. The result of blind experiment indicated that the results obtained from the SBE-FP and RDB method were consistent completely. It was confirmed that this SBE-FP method can be used to genotype the 8 types of P thalassemia mutations precisely.3. ConclusionsIn this study, we identified a novel frameshift mutation of CDs 15/16(+G) in the p-globin gene, elucidated the relationship of its phenotype and genotype, analyzed its functional effect at the mRNA levels and determined the RFLP-haplotype linked to this mutation. This mutation was reported firstly in the world and enriched the mutation spectrum and genetic database of P thalassemia. Furthermore, the novel PTC mutation of CDsl5/16 (+G) in a p°-thalassaemia gene would be a valuable model for studying the mechanisms of the NMD. In addition, the established SBE-FP assay for simultaneous detection of the eight most common causative mutations of Chinese P thalassemia was a homogenous reaction system which based on the format of 384-well PCR high-tense plate. The results demonstrate that this high-throughput method is accurate, rapid and cost-effective and is suitable for large-scale gene screening for p thalassemia. In summary, this study provided some valuable genetic data and detection method for the genetic counseling, prenatal diagnosis and prevention program of large-scale population screening of p thalassemia.