Application Of DHPLC Assay In Mutations Analysis Of Two Common Birth Defects

Birth defects are defined as structural abnormalities present at birth. They account for approximately a third of all paediatric admissions and are the leading cause of infant mortality in the western world as well as in China. According to the information published by the Ministry of Health, the national overall incidence of birth defects is about 11‰and about 70%disability are caused by birth defects in 80 million disabled population in China. The results, about birth defects investigated by Tian PL et al. among 0-5 year-old children of Guangdong province in 2004, shown that congenital heart disease was the most common birth defects followed by thalassemias. Domestic large number of studies have manifested that congenital heart disease is one of the most common encountered heart disease during childhood, occurring in 6-8 per 1000 live births. Despite its prevalence and clinical significance, the aetiology of CHD remains largely unknown owing to the complexities of its genetic background and pathogenesis. In addition, the thalassemia is the most common single-gene diseases with the wide geographical spread and high carrying rate. Because of the serious economic and spiritual burden to society and the family from both of them, it is important to establish an effective technology for early screening and diagnosing to prevent congenital heart disease and the severe thalassemia occurring. It will be of great significance to reduce the incidence of these diseases and improve the quality of life in Chinese population.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 hetroduplexes 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 used denatured gradient gel electrophoresis (DGGE), chemical cleavage of mismatch (CCM), conformation sensitive gel electrophoresis (CSGE) and single-strand conformation polymorphisms (SSCP) technologies and has been widely used in mutational screening and molecular diagnosis of hereditary diseases. According to these adavantages of DHPLC assay, on the one hand, the purpose of this study is to establish a rapid, accurate and economic method for genotyping of the common nondeletionα-thalassemias and Hbα-variants in Chinese population, and on the other hand, we devote ourselves to studying on mutations of the predisposing genes of non-syndromic congenital heart disease in Southern China.Section one:Rapidly and accurately genotyping of common nondeletion a-thalassemias and Hb a-variants in Chinese by use of DHPLC assayObjectives:Nondeletion a-thalassemias, Hb a-variants, and in a few cases by both phenotypes represent a series of hemoglobinopathies as over 300 mutations have been identified for a-globin genes responsible for these diseases. A rapid and accurate genotyping method specific for mutations of a-globin genes is required for population screening as well as diagnosis of related diseases in both research and clinic setting.Methods:The samples were collected from different geographical regions in southern China where a-thalassaemias are highly prevalent. Human genomic DNA was extracted from leukocytes in peripheral blood by the standard phenol/chloroform method. Firstly, we analyzed a panel of unrelated samples from our previous mutational scanning by direct DNA sequencing to explore polymorphic variations and their population frequencies in entire sequences of a-globin genes from the cap site to the polyA addition site (α2-globin gene:107 samples, al-globin gene:58 samples). Secondly, we subjected 33 samples identified through direct DNA sequencing, carrying 22 known genotypes composed of ten mutations, were used as standard samples to establish experimental DHPLC conditions and DHPLC profile patterns corresponding to each genotype. Thirdly, for validation of the DHPLC assay, 171 samples (140 patients and 31 normal individuals) of various known genotypes were used to assess the sensitivity and the efficiency of the DHPLC system compared with RDB and/or direct sequencing analysis in a blind study.Results and conclusions:DHPLC assay was established based on analysis of 33 DNA samples from a group of various genotypes for ten mutations and three polymorphisms in a-globin genes. The reliability of this DHPLC assay was evaluated on 171 pre-typed samples by using RDB and/or direct DNA sequence analysis in a blind study. The results obtained by this assay were in complete concordance with the reference method. DHPLC was proved to be a simple, rapid, highly accurate, and cost-effective assay which can be used as a method for prenatal diagnosis and large scale population screening of common nondeletion a-thalassemias and Hb a-variants in Chinese.Section two:Study on mutations of the predisposing genes of non-syndromic congenital heart disease in southern China by DHPLC assayBackground and Objectives:Congenital heart disease (CHD) refers to malformation of the cardiovascular system that is present at or near the time of birth. These cardiovascular anomalies are common type of birth defect and a leading non-infectious cause of death in the first year of life, thereby making them an important form of cardiovascular disease in the young. 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. The pathogenesis of mutations within cardiac transcription factors have been increasingly recognized as genetic causes for CHD in humans. Many reports have implicated that there are obvious area-and race-specifics in the inherited factors during the development of polygenic disease. NKX2.5, independently described as a cardiac homebox gene has been identified approximately 29 missense and nonsense mutations in foreign population only three novel mutations were described in a familial ASD and a three bases insertion in a sporadic CHD until now. GATA4, a conserved zinc finger transcription factor, which mutation rate just 2%in foreign population, was found two novel mutations p.Val267Met and p.Val380Met in 31 CHD cases. Nine distinct mutations including one small deletion mutation, two small insertion mutations and six non-synonymous mutations were identified in 12 of the 486 patients by Weimin Zhang et al. These give a hint that the mutation loci and detection rate of transcription factors in Chinese population may be different from foreign population. Char syndrome is an autosomal dominant trait characterized by patent ductus arteriosus, facial dysmorphism and hand anomalies. Using a positional candidacy strategy, researchers mapped TFAP2B, encoding a transcription factor expressed in neural crest cells, to the Char syndrome critical region and identified 8 missense mutations in two foreign affected families. In the present study we performed a mutation screening in patients with non-syndromic congenital heart disease to further investigate the NKX2.5, GATA4 and TFAP2B genes and explore the correlation between genotypes and phenotypes in Southern China.Methods:The samples were collected from southern China. Human genomic DNA of 224 patients and 121 normal individuals were extracted from leukocytes in peripheral blood by the standard phenol/chloroform method. Whole coding regions of GATA4, NKX2.5 and TFAP2B genes were amplified by polymerase chain reactions (PCR). The PCR products were detected by DHPLC and samples with different melting profile shapes were sequenced, compared to GeneBank sequence databases.Results:1. A novel missense mutation c.788 C>G (GCC>GGC)inGATA4 gene coding region, was first identified in one patient with ventricular septal defect (VSD) compared with 121 healthy controls. The mutation resulted in the substitution of amino acid from alanine to glicine (p.Ala263Gly) and located in a highly conserved region of the N-terminal zinc finger. Three novel synonymous mutations were also detected (c.531C>A p.Ala177Ala, c.1308C>T p.His435His and c.1326G>A p.Ala422Ala). None of the controls found these mutations.2. There no mutations were found in NKX2.5 gene. Just three previous reported single nuclear polymorphisms (SNP)(rs2277923(c.63A>G GAA>GAG p.Glu21Glu), rs3729753 (c.606G>C CTG>CTC p.Leu202Leu) and rs703752 (c.975-61 G>T)) were detected. Both rs2277963 and rs3729753 were synonymous mutations. There were no differences between two groups of their alleles and genotypes frequencies.3. Two novel missense mutations of TFAP2B gene were first identified in sporadic non-syndromic patients with various phenotypes in Chinese population. One patient with totally endocardial cushion defect (ECD) carried the non-synonymous mutation c.31A>G (ATC>GTC, p.Ile11Val) and another six unrelated patients with different cardiac anomalies carried the same missense mutation (c.1006 G>A, p.va1336Ile).Conclusion:1. This study had shown that germ-lime missense mutation c.788 C>G of GATA4 gene may disrupt zinc coordination and may be highly associated with the ventricular septal defect. The p.Ala263Gly mutation affects a highly conserved amino acid localized N-terminal zinc finger. This changes the DNA binding affinity and the transactivation of downstream targets which results in impaired GATA4 expression and complex cardiac anomalies.2. None pathogenic mutations were identified among the patients, suggested that NKX2.5 gene mutations are relatively rare among CHD patients in Southern China. Thus, the diagnostic importance of NKX2.5 mutations may be confined to familial cases or specific subgroups of CHD phenotypes.3. Our datas suggest that TFAP2B can be a candidate gene for isolated non-syndromic CHD. Mutations in the gene encoding the TFAP2B transcription factor can cause Char syndrome with cardiac, craniofacial and hand abnormalities. However, TFAP2B mutations result in great phenotypic variability, which is believed to reflect different expression patterns of tissue-specific TFAP2 coactivators.