The Application Of Multiple Displacement Amplification In Single Cell Diagnosis Of Spinal Muscular Atrophy And β-thalassemia

β-thalassemia and Spinal muscular atrophy (SMA) are two common autosomal recessive diseases.β-thalassemia is one of the inherited autosomal recessive hemoglobinopathie diseases which is common in south China. About 2.8% of south Chinese people are homozygous ofβ-thalassemia. The disease cause by the mutation in human beta globin gene cluster, which located on chromosome 11 and includes 5 functional genes and 1 pseudo gene. Nearly 200 different mutations of been found in human beta globn gene cluster, and 28 different mutations in south china. The mutation of human beta globing gene cluter will result in anaemia and organ damage, leading to reduced the life expectancy. Spinal muscular atrophy is a fatal autosomal recessive, the incidence of which is 1/6,000 to 1/10,000 and the carrier frequency is 1/40 to 1/50. SMA is caused by major mutation in survival motor neuron gene 1 (smn1) gene located on chromosome 5q13. The mutation of smn1 gene will cause hypotonia and muscle weakness, and gradually affect the function of walk, stand, swallowing and even breathing and result in death. there still no effected theropy strategy for both the two diseases. prenatal diagnosis is the most common to prevent the birth of affect baby. but it need to terminnal the pregnancy when SMA is diagnosis, which may make great hurt of the paitent family.Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis. PGD involves in vitro fertilisation (IVF) , detection of genetic defects in polar body or in granulosa cell or in one or two blastomeres biopsied from embryos at about eight cell stage, and transfer of unaffected embryos to the uterus after genetic diagnosis using PCR, FISH, of other techniques. It can diagnosis the disease without termination of pregnancy when genetic defect was detected. There about 20,0000 cycles of PGD have been perfrom and more than 6,000 heathy baby have born accroding to the European Society of Human Reproduction and Embryology. PGD have bring hope for the couple that high risk in birth of an baby with genetic defect.There are many reports of PGD of SMA andβ-thalassemia.Signle strand conformation polymorphism (SSCP), denatured gradient gel electrophoresis (DGGE), fluorescenc PCR, nest PCR and minisequencing have been applied to the PGD ofβ-thalassemia, and restriction fragment length polymorphism (RFLP), allele-spcecific PCR, fluorescenc PCR and minisequencing have been used for PGD of SMA. All the methods above face to the problem that only a limited amount of DNA template ( one to two cell ) is avaliable for DNA analysis, and thus have some drawbacks: 1. limited marker can be detect; 2. DNA template would be exhaust after PGD, and no DNA template can be left for forther analysis; 3. Each reation for any diseases have to be carefully optimizate, which would cause a lot of time and no easy to estabilish; high allele drop out(ADO) rate and so on.To over come this problem , whole genome amplification (WGA) technique have been used for PGD as the initial step before DNA analysis to increate the amount of template. There are several WGA available for PDG, such as primer extension preamplification PCR (PEP-PCR), degenerate oligonucleotide primed PCR (DOP-PCR) and multiple displacement amplification (MDA). EP-PCR and DOP-PCR are all PCR-base WGA, and both have some drawbacks, such as imcomplete coverage, higt ADO rate, small size of production which do not suit for some downstream DNA analysis methods that need higt quanity DNA template. MDA is one WGA methods first descrip by prof. Lizardi. MDA is based on the ability of the Phi29 DNA polymerase, to cause strand displacement isothermal with random amplification initiation points by using) random primers. MDA have high processivity and fidelity and can generate long DNA fragment and large amont of DNA template. The aim of our experiment is to estabilish a protocol for single cell diagnosis of SMA andβ-thalassemia conbind with MDA. And lay a foundation for the PGD of SMA andβ-thalassemia.Objiective:The aim of this experiment is to estabilish a single cell diagnosis protocol for SMA andβ-thalassemia.We optimizated the incubation time of MDA by using AS-PCR, microstallite analysis and RDB to compare the amplification efficiency, ADO rate and the total amount of DNA product of different incubation time of MDA, and combind MDA, AS-PCR and microsatellite analysis to amplified the SMN gene from single cell and diagnosisβ-thalassemia by combind MDA and RDB.Materials:Five fibroblast cell lines were used for this experiment, including two SMA fibroblast cell lines with homozyous deletion of smn1 gene (S1 and S2); oneβ-thalassemia fibroblast cell line carried heterzyous mutation ofβ41-42/β17 (TH1); oneβ-thalassemia fibroblast cell line with homozygou mutation ofβ41-42/β41-42; one normal fibroblast. A total of 49 single fibroblast cell from 5 diffirent cell lines were collected. (13,10,8,10,8 single cell from TH1,TH2,S1,S2,N1 respectively).Methods:1. prepare the single cell2. single cell is amplify MDA.Each fibroblast cell lines divided into two group: 4 hour incubation group and 16 hour incubation group.3. amplify smn1 and smn2 gene from MDA product by using AS-PCR4. amplify two microstallite (D5S351, D5S435) from MDA product by using F-PCR5. amplify the human beta globin gene cluter from MDA product by using RDB Result:1. The total amplification efficiency of MDA is 89.3%(342/383), The amplification efficiency are 90.48% (19/21) for smn1;94.87%(37/39)for smn2;94.8% (37/39) for D5S351, 87.18% (34/39) for D5S435;92.02%(127/138)for MDA combind with STR and AS-PCR;88% (220/250) for RDB. The total ADO rate of MDA is 13.92%(11/79). The ADO rate is 16.22%(6/37)for D5S351; 12.5%(2/16) for D5S435;15.09%(8/35) for MDA combind with STR and AS-PCR .11.54%(3/26) for RDB.2. The amplification efficiency is 90.48%(171/189) and 88.14%(171/194) for 16h group and 4h group respectively(chi-square statistics, P>0.05). The ADO rate is 17.07%(7/41) and 10.53%(4/38) for 16h group and 4h group respectively (chi-square statistics,P>0.05). No significant difference was found in the amplification efficiency, ADO rate of MDA between 4 hour incubation group and 16 hour incubation group.Conclusion:1. The amplification efficiency, ADO rate is similar between 4h and 16h group.We can decrease the incubation time of MDA to 4h, and decrease the total time of single cell genetic diagnosis,which lay the foundation for the PGD.2. This is the first time to combind MDA, AS-PCR and microsatellite analysis to amplified the SMN gene from single cell. This method can diagnosis the homozygou deletion of Smn1 gene, which lay the foundation for PGD of SMA3. This is the first time to diagnosisβ-thalassemia by combind MDA and RDB. The method can detect 18 mutations ofβ-thalassemia in chinese population, which lay the foundation for the PGD ofβ-thalassemia.