Study On Rapid Prenatal Diagnosis Of Aneuploidies Involving Chromosome13, X And Y

Objective1To screen out polymorphic short tandem repeat (STR) loci on chromosome13, X and Y in Tianjin Han population of China and set up a method to diagnose chromosome aneuploidies and provide basic data for using STR loci in prenatal diagnosis of aneuploidies accurately and rapidly.2To establish a method for prenatal diagnosis of aneuploidies involving chromosome13, X and Y and discuss the clinical value of quantitative fluorescence polymerase chain reaction (QF-PCR) in rapid prenatal diagnosis.Methods1According to the literature we chose30loci on chromosome13, X and Y as candidate loci. QF-PCR and capillary electrophoresis was applied to350unrelated individuals of Tianjin Han population of China. The relevant data were analyzed by ABI Prism GeneMapper v3.0software. Allelic frequencies and genotype frequencies were calculated by direct counting. The frequencies of the genotypes were checked using Chi-square test to verify Hardy-Weinberg Equilibrium (P>0.05). Genetic polymorphisms data were calculated by PowerStatsV12software. The ratio scopes of heterozygote peak areas, the average peak areas ratio and95%confidence interval (CI) were calculated using SPSS17.0software. Two alleles of each locus were sequenced for confirmation of repeat sequence and repeat number.2Polymorphic loci located on chromosome13, X and Y were used to examine362peripheral blood samples and452prenatal diagnosis samples (including373cases of amniotic fluid and79cases of villi) by QF-PCR. The results were compared with karyotype analysis.Results1The polymorphisms of STR loci on chromosome13, X and Y1.1Based on preliminary experiments we finally chose D13S305, D13S631and D13S634markers on chromosome13, HPRTB, DXS6803and DXS6809markers on chromosome X, DYS19, DYS390and DYS393markers on chromosome Y for the loci of the present study.1.2The repeat sequence of D13S305, D13S631and D13S634locus was CTTT, ATCT and AAGA.11,7and11alleles were found for each locus. No significant deviations from the Hardy-Weinberg equilibrium were observed in these three STR markers (P>0.05). The He of these three STR loci was0.833,0.754,0.796. The Ho was0.809,0.714,0.757. The PIC was0.810,0.714,0.765. The PD was0.948,0.893,0.927. The PE was0.615,0.451,0.522.1.3The repeat sequence of HPRTB, DXS6803and DXS6809locus was TCTA, GATA and ATCT.10,6and10alleles were found for each locus. No significant deviations from the Hardy-Weinberg equilibrium were observed in these three STR markers (P>0.05). The He of these three STR loci was0.748,0.649,0.806. The Ho was0.607,0.700,0.713. The PIC was0.706,0.599,0.775. The PD was0.894,0.814,0.931. The PE was0.299,0.428,0.449.1.4The repeat sequence of DYS19, DYS390and DYS393locus was TAGA, TCTA and TATC.5,6and6alleles were found for each locus. The gene diversity (GD) values for each locus were0.743,0.731,0.634.1.5Combined six markers on chromosome13and X, the ratio scope of heterozygote peak areas was0.68～1.49. The average peak area ratio was1.09±0.18. The95%CI was1.08-1.10.2Genetic diagnosis and prenatal gene diagnosis of chromosome aneuploidy2.1The genetic diagnosis results362peripheral blood samples were successfully amplified.352samples gave a normal QF-PCR results and3cases of47,XXY,4cases of45,X,1case of47,XYY,1female with karyotype46,XY and1mosaic case were diagnosed correctly. Two mosaic cases failed to diagnose because of the low percentage of mosaicism.2.2The prenatal gene diagnosis results2.2.1Fetal sexing was successfully achieved in all cases and the results were in consistent with the chromosome karyotype. There were219male fetuses and233female fetuses.2.2.2There were20,12,12,13,35,35,37,19and18cases failed to amplify for each nine locus. The overall success rate was91.81%(415/452).2.2.3Combined six markers on chromosome13and X, the ratio scope of heterozygote peak areas was0.70～1.48. The average peak area ratio was1.12±0.18. The95%CI was1.10-1.14.2.2.41cases of trisomy13,1case of47,XXY and1case of45.X were detected by QF-PCR. Other412samples were normal and the results were in accordance with karyotype analysis.2.3The sensitivity and specificity of QF-PCR were86.67%(13/15) and100%(762/762).Conelusions1This research showed that D13S305, D13S631and D13S634markers on chromosome13, HPRTB, DXS6803and DXS6809markers on chromosome X, DYS19, DYS390and DYS393markers on chromosome Y were highly polymorphic in Tianjin Han population of China. They are good genetic markers on chromosome13,X and Y.2QF-PCR amplifying STR loci is a sensitive, specific, simple and rapid method for prenatal diagnosis of chromosome aneuploidies. It has a broad application prospect in clinic.