Molecular Genetic Analysis Of Rare Thalassemia In Three Chinese Families

α-thalassaemia most frequently results from deletion of one or bothαglobin genes from the chromosome 16.Occasionally,point mutations in critical regions of theαgenes may cause nondeletional a-thalassaemia.Very rarely,a-thalassaemia results from deletion of the remote regulatory elements（multispecies conserved sequences,MCS）,containing MCS-R1,MCS-R2,MCS-R3 and MCS-R4.At present,in all of these deletions,MCS-R2 is always removed and thus appears to be the major regulatory element which leads to almost complete down-regulation ofαgene expression.β-thalassaemia mostly results from point mutations ofβglobin gene,while occasionally is caused by large deletion ofβ-globin gene.Compound Heterozygosity forβ-thalassaemia and a-globin gene duplications,aggravating the imbalance ofαglobin chains andβglobin chains,may lead to severe intermediateβ-thalassemia.ObjectivesSome rare thalassemia types were easily mistaken by routine genes screening.Multiplex ligation-dependent probe amplification（MLPA）and array-based Comparative Genomic Hybridization（aCGH）were preferred for further detection to make definite diagnosis.Materials and methods1.Collected medical materials and blood samples of these three families;2.Extracted DNA from samples for the routine thalassemia gene diagnosis;3.Sanger sequencing and MLPA were used for further detection due to the negative results not corresponding with the haematological parameters.4.Gap-PCR was used to clarify the range and sequence due to the MLPA deletion frament5.aCGH was used to clarify the range due to the MLPA duplicated frament.Results1.In family 1,the proband was a 31-year-old woman who has the unexplain statement of microcytic hypochromic anemia.Routine thalassemia gene screening were negetive.MLPA was performed and showed a small heterozygous deletion of MCS-R2 with 4α-globin gene intact.Further investigation of deletion breakpoints showed that the 742-bp deletion upstream of theα-cluster removes the MCS-R2 element.The other four members in this family were inherited the same deletion.2.The proband in family 2 was a 6-month-old girl who carried severe HbH disease. Molecular analysis using Gap-PCR revealed onlyαSoutheast Asia（SEA）type of deletion(--^SEA),which was uncoordinated with the clinical features.MLPA showed a deletion of the MCS-R2 and SEA type of deletion,making the severe anemia explainable.The same deletion frament of 742-bp was proved by Gap-PCR.3.The proband in family 3 was a 2-year-old girl received regular transfusion at an interval of four weeks from the age of four month.Hematological parameters indicated severe thalassemia but DNA sequencing of theβglobin gene detected only a heterozygousβ-thalassemia mutation of IVS2-654（C>T）inherited by her mother.Her father was hematologically normal,howerver,his Gap-PCR result was the SEA type ofα-thalassemia.MLPA results found that the phenotype of her father was a compound heterozygosity forαduplication and Southeast Asia（SEA）type of deletion(αααα/--^SEA).The proband carried both the duplicatedαgene and mutationalβgene,leading to severe anemia.aCGH was performed and showed that theαduplicated CNV was 191 kb in size.Conclusion1.The deletion of MCS-R2（742bp）will lead toα⁰ thalassemia trait.2.It’s the first report that compound heterozygote of MCS-R2 deletion and α Southeast Asia（SEA）type of deletion will lead to severe HbH disease.3.There are no special characteristics among people withαduplication,for which is easily mistaken in the routine thalassemia gene sreening.4.When routine thalassemia gene screening shows only mutationalβgene among children with severe anemia,αgene duplications detection should be considered.