| Background and PurposeBirth defects are defined as abnormalities of morphology and physiological function or abnormalities caused by metabolic defect present at birth. With the development of modern society as well as the change of modern medical model, disease spectrum and death spectrum of human is undergoing a great change. Birth defects are gradually becoming the leading cause of infant mortality in China. The Prenatal and Postnatal Care Association investigated the birth defects of0～5year-old children of Guangdong province in2009, which showed that congenital heart disease was the most common birth defect in Guangdong. Large numbers of domestic studies have manifested that, accounting for approximately a third of all birth-defect diseases, congenital heart disease is the most common heart disease. Despite its prevalence and clinical significance, the aetiology of CHD remains largely unknown owing to the complexities of its genetic background and pathogenesis. Because of the serious economic and spiritual burden to society and the family from CHD, it is important to establish an effective technology for early screening and diagnosing to prevent congenital heart disease. It will be of great significance to reduce the incidence of CHD and improve the quality of life in Chinese population.Congenital heart disease (CHD) refers to malformation of the cardiovascular system present at or near the time of birth. These cardiovascular anomalies are common types of birth defect and a leading non-infectious cause of death in the first year of life. Although cardiac malformations may occur in the setting of multiple birth defects as part of a syndrome, most are found as isolated defects and are "non-syndromic". The etiology of CHD is multifactorial, with environmental and genetic factors playing important roles. Many reports have implicated that there are obvious area-and race-specifics in the inherited factors during the development of polygenic disease.Heart development is a complex event in embryonic development. The normal development of heart is regulated by the expression of many genes, which strictly follow the temporal and spatial order, thus the abnormality of anyone of these genes may cause congenital heart disease. Simple CHD is caused by many genes, which have different expression and regulation in different cardiac developmental stages and interact and collaborate with each other. The coordination and interaction with genes of LIM protein and zinc finger protein related to body development, energy metabolism, cell cycle, cytoskeleton, cell adhesion and cell migration in cardiac development form a ordered network system, which suggests that the heart is not a static organ, completely distinguishing children’s heart disease with adults’is wrong, and considering the heart’s development separately from the body’s other organs is unfair. From the beginning to the maturity and the function maintenance, the development of heart is a holistic and dynamic process.At present, the study of congenital heart disease is divided into four areas:ligand receptors such as NOTCH1, NODAL;transcription factors such as GATA4NKX2.5, TFAP2B, contractile proteins such as MYH1, ACTC, and other miscellaneous genes such as FLNA, ELN and so on. A host of studies in congenital heart disease have mainly concentrated on some critical transcription factors. However, other potent analysis on its genetic mechanism has been rarely reported. Heart development is affected by the regulation of multiple genes and their expression, in which strictly follow the temporal and spatial order. Therefore, any abnormality in quality or quantity of the genes involved may affect the heart development. Adapter protein is one of the proteins expressed in heart, but little is known about its role in the regulation on heart development. Adaptor proteins, composed of two or more proteins without enzymatic activity which interact with each other, regulate various cellular functions. Vinexin, CAP/ponsin, and SORBS2(ArgBP2) constitute a novel adaptor protein family. They have a novel conserved region homologous to the active peptide sorbin, as well as three SH3(src homology3)domains. A number of proteins binding to this adaptor family have been identified. CAP/ArgBP2/vinexin family proteins, adaptor proteins characterized by three SH3domains at their C-termini and a SoHo domain towards their N-termini, are known to regulate cell proliferation, cell adhesion and migration, cell apoptosis, cytoskeletal organization, and signaling conduction. SORBS2is expressed at high levels in the heart and is localized in the Z-bands of mature myofibrils.SORBS2, the other name is ArgBP2(Arg/Abl-binding protein, variant1), is a70kD protein that is expressed in non-muscle cells where it localizes in actin stress fibers and in the heart where it localizes in Z-bands. At least eight isoforms of human SORBS2ranging from492to1100amino acids have been derived. A single gene SORBS2located on4q35.1codes these isoforms, but multiple variants result from alternative splicing. Composed by23exons and22introns, variant1has a total length of226794bp, and cDNA is4939bp, encoding666amino acids.SORBS2has three C-terminal SH3domains that interact with c-Abl, a member of the Abelson family of non-receptor protein-tyrosine kinases, and an N-terminal sorbin homology (SoHo) domain that interacts with lipid raft proteins. The subcellular localization of this protein in epithelial and cardiac muscle cells suggests that it functions as an adapter protein to assemble signaling complexes in stress fibers, and that it is a potential link between Abl family kinases and the actin cytoskeleton. SORBS2can form complex with Abl and Cbl, thus promoting ubiquitination and degradation of Abl1or with AKT1and PAK1, thus mediating AKT1-mediated activation of PAK1. By inhibiting cell adhesion and cell migration, it inhibits tumor cell metastasis.SORBS2gene plays an important role in the stability of cytoskeleton. SORBS2is a scaffold protein that controls the balance between adhesion and motility by coordinating the function of multiple signaling pathways converging on the actin cytoskeleton. Microtubules, microfilaments, intermediate filaments, and its binding proteins constitute the cytoskeleton of the myocardial cells, which have an important effect on the maintenance of the normal form of myocardial cells, and regulate a range of physiological activities in the myocardial cells. Cytoskeletal components connect sarcomere and extracellular matrix in cells, which constitute a conductive loop of the contractile force to conduct contractile force and provide mechanical support. Myocardial cytoskeletal system not only contributes to the maintenance of myocardial cells’shape and the contractile activity, but also plays an important regulatory role in the anchoring and the function of membrane ion channels. Playing an important role in regulating the cardiac ion channels, cytoskeleton is a dynamic structure. The pathological process of myocardial ischemia, cardiac hypertrophy and heart failure include changes in the cytoskeleton. There is a great variety of cytoskeleton binding proteins with complex and unclear functions. The research on the relationship among cytoskeleton, skeleton binding protein and cardiac ion channel has important theoretical and practical significance in elucidating the mechanism of variety kinds of arrhythmia.SORBS2as a adaptin protein has multiple functions. SORBS2plays an important role in the signal transduction, assembly and maintenance of myofibrils in cardiomyocytes as well as in the regulation of actin cytoskeleton, thus it may contribute to the heart development, and further relate to the formation of congenital heart disease.Denaturing high performance liquid chromatography (DHPLC) had been proved to be a reliable, rapid, and highly sensitive method for detecting point mutations based on the detection of heteroduplexes in PCR products by ion pair reverse-phase HPLC under partially denaturing conditions. Its sensitivity and specificity can reach 96-100%, which are higher than commonly technologies and has been widely used in mutational screening and molecular diagnosis of hereditary diseases. In this study, we mainly use site-directed mutagenesis and gene cloning techniques to construct the known variations of SORBS2gene, and according to the exons and intron-exon junctions of SORBS2gene, primers are designed to establish denaturing high performance liquid chromatography analysis assay. And use this method to detect non-syndromic congenital heart disease patients in order to find related data of SORBS2gene in the Chinese population.According to advantages of DHPLC mentioned above, the purpose of this study is to establish a rapid, accurate and economic method to detect variations of the predisposing genes of non-syndromic congenital heart disease in Southern China.We intend to provide references for further research of the molecular mechanism of non-syndromic congenital heart disease, the clinical diagnosis, prevention and treatment of such diseases and the molecular mechanism research in other abnormal adaptor protein diseases caused by SORBS2gene variations.Samples and Methods1. SamplesA total of209Chinese patients (121male,84female, and4unkonw,0～6months) with CHD and120normal controls were recruited mostly from Boai hospital of ZhongShan and ZhuJiang Hospital and so on (PR China) for the study. Genomic DNA was extracted from2ml EDTA-anticoagulated peripheral blood by the standard phenol/chloroform method.2. Molecular analysisIn this study, we developed a PCR/DHPLC assay to detect SORBS2gene mutations. We used site-directed mutagenesis to construct the clone libraries containing13known variations of SORBS2gene. Firstly, the target gene fragments were obtained directly from human genome DNA samples using site-directed mutagenesis PCR method. Then these fragments were cloned into pMD20-T vector with gene engineering techniques. The host bacterial strain is E. coli DH5α. Each of inserted DNA fragment in these recombinants was sequenced to verify the DNA sequence of mutants cloned.For the exons and intron-exon junctions of SORBS2gene, primers were designed to establish DHPLC analysis assay. All23exons and intron-exon junctions of SORBS2gene were amplified in21reactions.3. Statistical analysisThe SORBS2gene denaturing high performance liquid chromatography assay detected13known variations of SORBS2gene repeatedly. The reproducibility of DHPLC analysis for detection of the13known variations of SORBS2gene was assessed by examination of the retention time and coefficients of variation.4. SORBS2gene mutation screeningThe collected genomics DNA samples used as templates for PCR amplification, the PCR products subjected to DHPLC analysis.5. Comprehensive analysis of the experimental results.ResultsMegaprimer PCR site-directed mutagenesis method was used to construct SORBS2gene known variations, the fragments of which were cloned into pMD20-T vector before sequencing. All clones were identified as desired variations, and the success rate of mutagenesis could reach100%.The denaturing high performance liquid chromatography analysis assay for SORBS2gene can rapidly and accurately distinguish the sequence variation of known variations from wild-type sequence.We collected209samples with CHD and120normal controls, none of the13known SORBS2gene variations was found in any patient, but4known single nucleotide polymorphism (SNP) and2novel variations in introns were detected.DisscussionDenaturing high performance liquid chromatography analysis is a recently developed method for mutation scanning. It is not limited by the type and the site of mutation, sequence-specific probe and purification are unnecessary, and run directly to detect genotype of sample after PCR amplification. Because of its simple, quick, low cost, accurate and the sensitivity, denaturing high performance liquid chromatography analysis method has been wide concerned.Established for SORBS2gene in this study, the denaturing high performance liquid chromatography assay can distinguish the known variations of SORBS2gene from wild-type rapidly and exactly. This method based on PCR/DHPLC is a high-throughput, highly sensitive, automated, rapid, accurate and economical mutation detection method.In recent years, the function of the adaptor protein family has been discovered continuously, among which SORBS2express abundantly in the heart. So it is considered to be related to the formation of congenital heart disease. However, disease-causing mutation of the SORBS2gene has not yet been reported, nor the study of SORBS2gene mutation screening. Denaturing high performance liquid chromatographic analysis was used to screen mutation with Chinese cases of congenital heart disease specimens in this study, but no novel mutation was found, which showed that, the carrying rate of SORBS2gene mutation is relatively low in China.The genetic defect in most patients with non-syndromic congenital heart disease (CHD) is unknown, although more than40different genes have already been implicated. Only a minority of CHD seems to be due to monogenetic mutations, and the majority occurs sporadically.At present, the mutation of transcription factor is considered to be the main cause of congenital heart disease, few reports refer to the analysis of other genetic etiology. Retrieving related literature on PubMed and other literature database, report about that SORBS2gene variations cause congenital heart disease in Chinese population was not found. Therefore, the genetic etiology of congenital heart disease in this study is of great significance and value.We hypothesize that SORBS2may contribute to a variety of acquired heart disease of cardiomyopathy and cytoskeleton to a certain extent, but have small effect on congenital heart disease. This will be our subsequent one of the important research direction.This study established a low-cost, high-throughput, rapid, and highly sensitive method for detecting SORBS2gene variations,which provided important references for further clarifying the molecular mechanism of the CHD, the clinical diagnosis, prevention and treatment of such diseases as well as research in the molecular mechanism of other abnormal adaptor protein diseases caused by SORBS2gene mutations. |
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