Identification Of A Novel Silent β Thalassemia Gene

Background and destination:Theβ-thalassemia is a fatal hemoglobin disease which is result from unbalancesynthesis of globin, and it is also one of the most common single gene disorders in theworld. The carrier rate ofβ-thalassemia in the word was found recently to be 4.83ï¼…by the newest research data. In China, the provinces located in the south of theYangtze River have a relatively high incidence ofβ-thalassemia, especiallyGuangdong, Guangxi, Hainan. The Guangdong Procince was found recently to be11ï¼…for the total carrier rate ofαandβthalassemia gene frequency. From 1949 theHBS was found and the "molecular disease" was present by Pauling, In the past 50years The research enclose the molecular pathology of hemoglobin disease give usmany classic theory for understanding the human gene and the eukaryotic geneexpression and regulation. In the recently years, as the accomplishment of the HumanGene Project, by the guidance of the theory of the genomic research, many researchresults about new thalassmia gene mutation given new understanding for us to learnthe theory of thalassemia pathogenesis and it is also enriching the mutationalspectrum of the human hemoglobin gene by the same time. Identification of manymutations especially these mutations located in the conserve region such as promoterregion, 5' UTR and 3' UTR etc. and the research about the transcriptional and thetranslation level of the globin (especiallyβ-globin) extending from these mutations.All these research are not only reveal the understanding of theβ-globin geneexpression regulation but also enrich the etiology of the hemoglobin disease andpresent the new theory for us to understand the possible pathogenesis of humangenetic diseases. Thalassemia intermedia is a group of thalassmeia with the intermediate clinicalmanifestation between thelassemia major and thalassemia trait. There is a high degreeof genetic heterogeneity about the clinical manifestation and the genetic deficiencybackground of thelassemia intermedia. The received diagnostic criteria of thethalassemia intermedia in the word is as followed: the Hb level is between 6-10g/L;the red blood cells parameter and the Hb electrophoresis results according withcharacter of thalassemia; the patient can live without blood transfusion; the patienthas a obvious thalassemia sings and symptoms. The possible genetic deficiency ofthis type thalassemia has been elucidated in the high incidence district of Europeanbased on the classic genetics concept. But there are still many unknown genedeficiency to be detected. In china, there is few reseach date of this type thalassemiahave been reported, and the systematic researches about the mutation spectrum andthe variation regular pattern of this special thalassemia have not been carried out. Thissituation is seriously hampering the progress of the clinic diagnosis, therapy,pregnancy diagnosis and genetic consulting in southern China.There are four conserved sequences in the promoter region of theβ-globin gene.These motifs inβ-globin gene include two CACCC boxes at nt-105 to nt-101 and-90 to -86 from the cap site, the CCAAT box at -76 to -72 and the TATA box at -30to -26. There are many research have been done about these conserve motifs and therelationship of their coactions with the trans-act factor. From the past years, theresearch data of the related type globin gene such asα-globin,γ-globin andδ-globinbased on the gel blockage, methylation interference and saturation mutagenesisexperiments have presented that most mutations degrade the avidity between theconserved sequences and the transcriptional factors followed the low efficiency of thegene transcription and the reduction of mRNA synthesis, thus causeβ⁺ orβ⁺⁺phenotype. In the past 30 years, many new thalassemia mutation have been elucidatedcontributed from the understanding of the molecular mechanism of thalassemia andthe progress of clinic diagnosis technology, most importantly, the large-scalepopulation carder screening of thalassemia has been effective carded out. In theseidentified mutations, 37 mutations belong toβ-thalassemia (including mutations toresult in the degrade of transcription, the incorrect RNA processing and the falsetranscription). The mutations located in the promoter region ofβ-globin gene were identified as a group of mutations which cause the degrade of transcription (usually tobe some type of base replacement in these conserve sequences). Including themutation identified in our research, there are 21 mutations in the promoter region ofβ-globin gene have been found.In our research, we got a classic thelassemia family, from the genotyping of thefamily members, we describe a novelβ⁺⁺-thalassemia mutation of-73 A＞T, which isthe only one currently found within the conserved CCAAT box at position -76 to -72from the Cap site ofβ-globin gene.Materials and methodsThe proband, an eight year old Chinese boy was diagnosed as thelassemiaintermedia, had mild splenomegaly and mild hemolytic facies, but has been nevertransfused since he was first examined at the age of four years for anemia. Accordingto the results of complete blood count and hemoglobin electrophoresis analysis, weconfirmed that the proband's father have a trait of microcytic hypochromic, i.e. withreduced mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH)(that are, 62.8fL/19.3pg) and an elevated level of HbA₂ (5.3ï¼…). The proband'smother and mother's two brothers have been identified as mildβ-thalassemiaphenotype, borderline or even normal MCV and MCH (that are 79.8fL/28.4pg,89.1fL/28.0pg, 90.0fL/28.1pg, respectively ), slightly elevated HbA₂ (3.84ï¼…, 3.95ï¼…,3.76ï¼…, respectively)β-Globin gene mutation analysis for the proband and his parentwas performed by using PCR-based reverse dot blot (RDB) hybridizationtechnique.After a screening of 24 known mutations in Chinese population, only onemutation of codons (CDs) 41-42 (-TCTT) was determined in the proband, which isinherited from his father, but the mutation inherited from the proband's mother hasnot been identified yet.In order to evaluate the functional effect of this novel mutation, real-timequantitative RT-PCR assay and relative standard curve method were used to analyzethe relative levels ofβ-globin mRNA of the 3 novel mutation carriers includingproband's mother and two maternal uncles.In addition we performed the haplotype analysis of 7 classical polymorphicrestriction enzyme sites by using PCR-based RFLP (restriction fragment length polymorphism) method and family linkage study. The RFLP haplotypes of theproband's twoβthalassemia chromosomes were determined.For diagnosis of the new mutation by the newest mutation detection technology, wedesigned and optimized the PCR amplification primers including the mutation areaand the DHPLC (Denaturing High Performance Liquid Chromatography) detectionmethod was used to analysis the genotype of the mutation carriers.Results and discussionDNA sequencing analysis of the proband and his family members identified amutation, a single nucleotide substitution of A＞T in the promoter region CCAAT box(-73) ofβ-globin gene Sequencing of the antisence strands did confirm thissubstitution mutation. By searching the literatures in PubMed and the online databaseof human haemoglobin variants and thalassaemias in the website of the Globin GeneServer (http://globin.cse.psu.edu/), it was confirmed to be a novelβthalassemiamutation, which was unreported previously. In summary, the proband is a compoundheterozygousβthalassemia patient. The causative mutations on his twoβthalassemiachromosomes are: the deletion mutation of CDs41-42(-TCTT), which is inheritedfrom his father and the substitution mutation of-73 A＞T, which is inherited from hismother, respectively. It is obvious that the genetic pattern ofβthalassemia gene ofthis family belongs to classical autosomal recessive inheritance.For it is the first time about natural mutation ofβ-globin conserved region CCAATbox at -76—-72 has been reported in the world. We consider that the new mutationshould beβ⁺ orβ⁺⁺ type based on the publication data of the two CACCC box atnt-105—-101, CACCC box at nt -90—-86 and TATA box at nt -30—-26 in theβ-globin gene promoter region and the induction of mutation results theγ-globingene transcription depressed data. Followed the two-step real-time PCR based ondouble standard curve confirmed that the new mutation result the degrading ofβ-globin gene expression to be 19.35ï¼…correspondent to the normal population. Sothe -73 A＞T mutation is aβ⁺⁺ thalassemia.By using PCR-based RFLP method and family linkage study, the haplotypeslinked to the -73 A＞T mutation and CD41/42(-TCTT) were identified as "+----+-" and "+----++", respectively. By the DHPLC analysis of the new mutation area, the characteristic eluting peakhas been obtained. With this result the gene diagnosis of the -73 A＞T carriers can becarry out in the future.The -73 A＞T mutation is the first one that has been found in the CCAAT box ofβ-globin gene promoter region. The founding enrich the mutation spectrum of thepopulation in southern China, and present the new genetic knowledge forunderstanding the genetic heterogeneity of thalassemia intermedia and the etiology ofhuman genetic disease. And it also can be used as a natural model to study thetranscription regulation mechanism ofβ-globin gene.