| Aims:Hypoplastic Right Heart Syndrome (HRHS) is one complex Congenital Heart Disease (CHD) characterized by different degrees of hypoplastic right ventricle, tricuspid valves and/or pulmonary valves(valve stenosis or atresia); Second Heart Field (SHF) supplies cells for right ventricular development, and SHF related genes regulate development of outflow tract and right ventricle during embryogenesis. Genetic engineering animal model suggests that SHF transcription factor knockout animals have similar cardiac phenotype to human HRHS; the hypothesis of our experiment is that SHF gene mutation may cause occurence of HRHS; so we plan to investigate the association between variants in SHF related genes and HRHS by next generation sequencing for candidate genes.Methods:31patients suffered from HRHS as case group and383patients with isolate coronary artery disease (Structural heart diseases were excluded by echocardiography) as control group were enrolled from our hospital. DNA was extracted from peripheral blood. Full length of17genes like Hand2, Smyd1, Mef2c, Tbx20, Foxh1, Islet1, Gata4, Gata5, Gata6, Nkx2-5, Foxc1, Foxc2, Foxa2, Tbx1, Shh, Fgf8, Fgf10were chosen for PCR in first16DNA samples from HRHS patients. The PCR productions were used for Ion Torrent sequencing. Then Data from sequence were compared with information of1092normal persons from1000genomes project and bioinformatics analyses were done. Single nucleotide variants (SNV) were selected from Ion Torrent sequence data for further validation by sanger sequence. The validated SNVs were then detected in31HRHS cases and383controls by Sequenom Mass ARRAY(?) SNP genotyping.Results:The functional regions like exon, promotor, enhancer binding cite,3’UTR and5’ UTR were chosen as a unit for bioinformatics analyses. The affected rates of low frequency variants in functional region of10genes such as Foxa2, Nkx2-5, Hand2, Tbx1, Fgf8, Isl-1, Foxc2, Gata6, Mef2c, Tbx20in HRHS group were significantly higher than in1000genomes group(p<0.05). Among of them, low frequency variants in Foxa2(p=3.16×10-16),Nkx2-5(p=2.91×10-8), Hand2(p=2.48×10-6), Tbx1(p=2.48×10-6), Fgf8(p=1.96×10-4), Isl-1(p=1.96×10-4), Foxc2(p=0.014), Gata6(p=0.014) were absent in1000Genomes controls. Low frequency variants in Mef2c functional region could increase HRHS risk to22-fold compared to no low frequency variants group (OR=23.49,95%CI=8.27-66.69, p=7.8×10-15), while low frequency variants in Tbx20could increase HRHS risk to15-fold (OR=16.54,95%CI=4.27-64.11, p=8.3×10"6).19SNVs were selected from sequencing data and were confirmed in corresponding HRHS samples by sanger sequence. The further Sequenom MassARRAY(?)SNP genotyping revealed that in common variant Gata5:rs6061550C>T, if the wild type CC and heterozygote CT as reference group, subjects carrying TT variants had a significant5.51-fold higher risk of HRHS; For common variant Foxc2rs34221221A>G, if homozygote genotype AA was used as reference, subjects carrying mutant homozygote GG had a significant2.6-fold increased risk of HRHS.Conclusion:The low frequency variants and common variants both play important roles in occurence and development of HRHS. Low frequency variants in functional region of10genes such as Foxa2, Nkx2-5, Hand2, Tbx1, Fgf8, Isl-1, Foxc2, Gata6, Mef2c, Tbx20could increase the risk of HRHS significantly; while common variants in Gata5and Foxc2could increase risk of HRHS too. We infer several probabilities for HRHS:One is susceptible common variants lead to occurrence of HRHS under special circumstances; another is low frequency variants in SHF genes disturbing pathway regulation cause HRHS; The last one is mosaic model composed by low frequency variants and common variants, on the basis of common variants increasing susceptibility, low frequency variants lead to the occurence of HRHS. |
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