Single nucleotide polymorphism of NKX2-5 gene with sporadic congenital heart disease in Chinese Bai population

Background: Congenital heart disease (CHD) is the most common birth abnormality, especially for sporadic CHD. However, the etiology of sporadic CHD is largely unknown. NKX2-5, the earliest sign of cardiac progenitor cell differentiation, plays a key role in cardiac morphogenesis, and the mutation of this gene can cause sporadic CHD. Purpose: To investigate the association of genetic variations of NKX2-5 with sporadic CHD in Chinese Bai people. Methods: The whole 2 coding exons and flanking intron sequences of NKX2-5 gene were screened using DNA sequencing in 70 Chinese Bai patients with sporadic CHD and 136 healthy controls. Results: A novel heterozygous DNA sequence variant (DSV), 1433A>G, was identified in one tetralogy of Fallot (TOF) patient and one persistent left superior vena cava (PLSVC) patient, but none in controls. The frequency of single nucleotide polymorphism (SNP) rs2277923 in CHD group was significantly higher than that in control group. The allele and genotype were associated with the occurrence of CHD. Conclusion: The novel DSV (1433A>G) may be relevant with TOF and PLSVC, and the SNP rs2277923 of NKX2-5 gene contributes to the risk of sporadic CHD in Chinese Bai people.     

Somatic mutations in NKX2–5, GATA4, and HAND1 are not a common cause of tetralogy of Fallot or hypoplastic left heart

The majority of congenital heart disease (CHD) occurs as a sporadic finding, with a minority of cases associated with a known genetic abnormality. Combinations of genetic and environmental factors are implicated, with the recent and intriguing hypothesis that an apparently high rate of somatic mutations might explain some sporadic CHD. We used samples of right ventricular myocardium from patients undergoing surgical repair of tetralogy of Fallot (TOF) and hypoplastic left heart (HLH) to examine the incidence of somatic mutation in cardiac tissue. TOF is a common form of cyanotic CHD, occurring in 3.3 per 10,000 live births. HLH is a rare defect in which the left side of the heart is severely under-developed. Both are severe malformations whose genetic etiology is largely unknown. We carried out direct sequence analysis of the NKX2–5 and GATA4 genes from fresh frozen cardiac tissues and matched blood samples of nine TOF patients. Analysis of NKX2–5, GATA4, and HAND1 was performed from cardiac tissue of 24 HLH patients and three matched blood samples. No somatic or germline mutations were identified in the TOF or HLH patients. Although limited by sample size, our study suggests that somatic mutations in NKX2–5 and GATA4 are not a common cause of isolated TOF or HLH.     

NKX2.5基因突变在先天性心脏病发病机制中的研究进展

摘要：NKX2.5/CSX基因的突变造成多种形态的先天性心脏病（congenital heart disease CHD）,是目前研究最多的与心脏发育密切相关的转录因子基因之一。 NKX2.5基因的生殖细胞突变,目前已经发现29种,分别为无义突变、错义突变、缺失等,导致NKX2.5蛋白与DNA结合功能下降或失活,在先心病发病中占据重要位置。最近研究发现,在复杂型先心病中存在多种NKX2.5基因的体细胞突变,多个体细胞突变可能在先心病发病中起叠加效应。     

Optimization of the Affymetrix GeneChip Mapping 10K 2.0 Assay for routine clinical use on formalin-fixed paraffin-embedded tissues.

The use of chromosomal copy number changes as markers for tumor behavior or as prognostic markers for patient outcome has been suggested. However, current clinically used technologies cannot perform genome-wide assessment of chromosome copy number and analysis of loss of heterozygosity in the same assay for paraffin-embedded tissue. We have optimized the Affymetrix GeneChip Mapping Assay for the 10K 2.0 array for use with formalin-fixed, paraffin-embedded (FFPE) tissues. This technology uses single nucleotide polymorphism (SNP) arrays to assess the changes in chromosomal copy number and loss of heterozygosity. DNA from 3 paired tumor/normal samples of adrenal tumors and 4 samples of renal tumors were processed with modifications to the manufacturer's protocol. Modifications at different steps were evaluated for their effects on SNP signal-detection and call rates. Frozen samples showed 99.6%+/-0.3% signal-detection rates and 94.7%+/-3.0% SNP call rates. FFPE samples labeled with the original protocol failed to produce enough polymerase chain reaction products for hybridization, whereas the same samples processed with the optimized protocol had signal-detection rates of 97.4%+/-0.018% and SNP call rates of 90.9%+/-0.034%. The average SNP call concordance between fresh and matching FFPE samples was 96%. Chromosomal aberrations detected in the frozen tumors were also detected in the FFPE tissues. Our optimized protocol significantly improves the performance of the FFPE samples in the Affymetrix GeneMapping Assay with the 10K 2.0 SNP array. This optimized protocol opens up the potential for the GeneChip Mapping assay to be used in the development of clinical test assays.     

A novel CSX/NKX2-5 mutation causes autosomal-dominant AV block: are atrial fibrillation and syncopes part of the phenotype?

The prevalence of congenital heart defects is approximately 1% of all live births. Identifying the genes responsible for cardiac malformation is the first step to understand pathogenesis. Heterozygous mutations in the CSX/NKX2-5 (NKX2E) gene have been identified to cause atrial septal defect (ASD) and/or atrioventricular (AV) conduction disturbance in some families. However, there is great variability in expressivity of the phenotype between the patients with a CSX/NKX2-5 mutation.We screened four sporadic patients and three index cases of families with ASD and/or conduction defects. In one of them, a CSX/NKX2-5 mutation was identified. This novel mutation (p.Tyr256X) was inherited in a three-generation family causing five individuals to have cardiac anomalies ranging from ASD to arrhythmias. Interestingly, all the observed AV conduction disturbances were at the nodal level, manifesting first as an AV block of the first degree and evolving toward a second-degree block. Atrial fibrillation, previously reported in three individuals with CSX/NKX2-5 mutations, was observed in three patients.     

Application of array CGH on archival formalin-fixed paraffin-embedded tissues including small numbers of microdissected cells

Array-based comparative genomic hybridisation (aCGH) has diverse applications in cancer gene discovery and translational research. Currently, aCGH is performed primarily using high molecular weight DNA samples and its application to formalin-fixed and paraffin-embedded (FFPE) tissues remains to be established. To explore how aCGH can be reliably applied to archival FFPE tissues and whether it is possible to apply aCGH to small numbers of cells microdissected from FFPE tissue sections, we have systematically performed aCGH on 15 pairs of matched frozen and FFPE astrocytic tumour tissues using a well-established in-house human 1 Mb BAC/PAC genomic array. By spiking tumour DNA with normal DNA, we demonstrated that at least 70% of tumour DNA was required for reliable aCGH analysis. Using aCGH data from frozen tissue as a reference, it was found that only FFPE astrocytic tumour tissues that supported PCR amplification of >300 bp DNA fragment provided high quality, reproducible aCGH data. The presence of necrosis in a tissue specimen had an adverse effect on the quality of aCGH, while fixation in formalin for up to 96 h of fresh tissue did not appear to affect the quality of the result. As little as 10-20 ng DNA from frozen or FFPE tissues could be readily used for aCGH analysis following whole genome amplification (WGA). Furthermore, as few as 2000 microdissected cells from haematoxylin-stained slides of archival FFPE tissues could be successfully used for aCGH investigations when WGA was used. By careful assessment of DNA integrity and review of histology, to exclude necrosis and select specimens with a high proportion of tumour cells, it is feasible to preselect archival FFPE tissues adequate for aCGH analysis. With the help of microdissection and WGA, it is also possible to apply aCGH to histologically defined lesions, such as carcinoma in situ.     

Whole genome SNP arrays using DNA derived from formalin-fixed, paraffin-embedded ovarian tumor tissue

Array-based genotyping platforms, such as the Affymetrix mapping array, have been validated as reliable methods for obtaining high-resolution copy number and allele status information when using DNA derived from fresh tissue sources. However, the suitability of such systems for the examination of DNA derived from formalin-fixed, paraffin-embedded (FFPE) tissues has not been tested. Therefore, we analyzed DNA derived from five matching fresh frozen and FFPE ovarian tumors for gene copy number changes and loss of heterozygosity using the Affymetrix GeneChip Human Mapping 10 K Array Xba 131. The data was analyzed using Affymetrix proprietary software, GeneChip DNA Analysis Software, and Chromosome Copy Number Tool. The average SNP call rate (rate of successful genotype identification) of the fresh samples was 89.44% (range 78.72-96.22%, median 92.72%) and was only slightly lower for the matching FFPE samples at 83.48% (range 76.93-93.17%, median 82.60%). The average concordance (rate of agreement between successful genotype calls) between the fresh and matching FFPE samples was 97.06% (range 92.70-99.41%, median 97.52%). Loss of heterozygosity (LOH) profiles of the fresh and FFPE samples were essentially identical across all chromosomes. Copy number data was also comparable, although the quantification of copy number changes appears overstated in the FFPE samples. In conclusion, we have shown that it is possible to achieve high-performance outcomes using FFPE-derived DNA in the Affymetrix 10 K mapping array. This advance will open up vast archival tissue resources to high-resolution genetic analysis and unlock a wealth of biological information.