Gene Mutation And CNVs Analysis For Congenital Heart Defects With Limb Anomalies

SECTION1:Congenital Heart Defects with Limb Anomalies Related Genes Mutation Detection and Functional AnalysisBackground:Congenital heart defects are the most common birth defect. Limb deformities are the most common extracardiac malformation which is considered as the second most common birth defect. Among all the syndromes of cardiac malformations, congenital heart defects and limb deformities often occur in combination, especially upper limb deformities. More than100syndromes including the heart and upper limb malformations have been reported. The known genes associated with heart and/or limb deformities include NKX2-5（RefSeq:NM₀04387.3）, GATA4（RefSeq:NM₀02052.3）, TBX5（RefSeq:NM₀00192.3） and SALL4（RefSeq:NM₀20436.3）Objective:To investigate the mutation frequencies of four candidate genes TBX5, NKX2-5, GATA4, and SALL4and functional analysis.Methods:Twenty-three congenital heart defect patients with limb anomalies were recruited in the Department of Cardiothoracic Surgery of Second Xiangya Hospital from Sep2009to Mar2012, which included sixteen congenital heart defect patients with upper limb anomalies, four congenital heart defect patients with lower limb anomalies and three congenital heart defect patients with both upper and lower limb anomalies. All patients underwent detailed clinical examination including transthoracic echocardiography and ECG, and a part of patients underwent limb X-ray examination and abdominal ultrasound examination. DNA of all patients and some patients’parents was extracted from the peripheral blood for PCR amplification, then TBX5, NKX2-5, GATA4, and SALL4gene sequencing and functional prediction analysis.Results:By X-ray of the defective limbs, defects in skeletal deformities of the limbs were identified. By mutation analysis, a novel missense mutation of NKX2-5c.257T>C （p.F86S） was detected in a patient diagnosed as partial atrioventricular septal defect associated with polydactyly （24digits）. Her father who carried a missense mutation c.257T>C （p.F86S） and synonymous mutation c.186T>A （p.A62A） was phenotypically normal. And her mother bears no NKX2-5mutation.Conclusions:Missense mutation of NKX2-5c.257T>C （p.F86S） could lead to congenital heart defect, partial atrioventricular septum defects. SECTION2:Investigation of CNVs Associated with Congenital Heart Defects with Limb AnomaliesBcakgroud:Copy number variations （CNVs） is known to be associated with congenital heart defects. And it is indicated that pathogenic CNVs more frequently associated with congenital heart defect with extracardiac defects. But now there is no investigation of CNV with congenital heart defects with limb anomalies.Objective:To investigate the relationship between the pathogenic CNVs and congenital heart defects with limb deformities, discover the novel pathogenic CNVs, and ascertain the new candidate genes associated with congenital heart defects with limb deformities.Methods:Nineteen out of twenty-three congenital heart defect patients with limb anomalies were selected in the Department of Cardiac Surgery of Second Xiangya Hospital from Sep2009to Mar2012, which included twelve congenital heart defect patients with upper limb deformities, four congenital heart defect patients with lower limb deformities, and three congenital heart defect patients with both upper and lower limb deformities. In order to seek CNVs regions related to the heart-limb deformities, whole genome CNVs were analyzed in all patients and some patients’parents via Array-SNP chip technology. Results:Three pathogenic CNVs were found in the three congenital heart disease patients with limb anomalies, which were chromosome3p25.2microduplication,16p13.3microduplication and22q11.21microdeletion respectively.Conclusions:1. Microduplication of3p25.2encompassing RAF1associated with congenital heart disease suggestive of Noonan syndrome.2. Our study extends the phenotypic spectrum of the16p13.3microduplication syndrome and increases the phenotype of diaphragmatic eventration and urine incontinence.