Rapid, Accurate Detection Of TMPRSS6 Gene Mutations With High-resolution Melting Analysis

Background and PurposeIron is the most abundant essential element which present in all cells in human body. Its main functions include the hemoglobin synthesis and oxygen delivery, but also involved in a number of biochemical processes in the body which including the mitochondrial electron transport, catecholamine metabolism and DNA synthesis. In addition, about half of the enzymes and coenzymes in the tricarboxylic acid cycle contain iron or need the presence of iron. Therefore, many disorders caused by the destruction of balance of iron metabolism in human body. Generally, the iron content in a narrow range in human body, iron deficiency and iron overload can cause the corresponding diseases. Iron deficiency can cause iron deficiency anemia (IDA) and neurological diseases of children, whereas iron overload can cause hemochromatosis. Iron deficiency affects over half a billion people worldwide, the most common disease as the iron deficiency anemia. There is a type of iron deficiency anemia known as iron-refractory iron deficiency anemia (IRIDA). The key features of IRIDA are:low mean corpuscular volume (MCV), low mean corpuscular hemoglobin (MCH), low transferrin saturation, abnormal iron absorption with no hematological improvement following treatment with oral iron and sluggish iron utilization. Some scholars have found that the disease is an autosomal recessive disease, which the patient transmembrane serine protease 6 (TMPRSS6 or named as Matriptase-2) gene mutation/mutations cause increased levels of serum hepcidin.Transmembrane serine protease 6 is a typeⅡmembrane serine protease, cloned the first generation and mapped in 2005. Some scholars believe that TMPRSS6 is closely related to iron metabolism balance in the human body.Transmembrane serine protease 6 regulates iron homeostasis by controlling the level of hepcidin in our body. Hepcidin is a hormone produced by the liver that can inhibit absorption of dietary iron in the intestinal tract and the release of iron from macrophages. Hepcidin binding the iron release channel membrane protein, internalizated and hydrolyzed in the lysosomes, thereby preventing iron release into the serum from the digestive tract cells and macrophages. Hepcidin increased levels in the human body can reduce serum iron content, leading to iron deficiency anemia. Hepcidin reduced expression can increase serum iron content, resulting in iron overload in human body.Transmembrane serine protease 6 encoding gene TMPRSS6 is located on 22q12.3. The TMPRSS6 gene mutations that cause the iron deficiency anemia have been reported in 2008. So far, foreign countries have a set of TMPRSS6 gene mutations causes iron-refractory iron deficiency anemia were reported. Meanwhile, some scholars believe that TMPRSS6 gene is also linked to iron overload. Because of the genetic differences, there are differences in the TMPRSS6 gene mutations between Chinese populations and other peoples. According to the search results in literature search databases such as PubMed, the TMPRSS6 gene mutations that cause the iron deficiency anemia were not reported in Chinese population. According to the researches, there is a high proportion in our children with iron deficiency.High-resolution melting analysis is a PCR-based method for detecting DNA sequence variation by measuring differences in the melting of DNA duplexes. The melting profile of the PCR product depends on its length, GC content and GC distribution. When the fragment under certain conditions, single base changes have an impact in the melting temperature of the fragment, and it can be detected through the high resolution and sensitivity detection technology. A homozygous sample after PCR amplification, contains a homoduplex, G:C and A:T single base changes have an impact in the melting temperature of the fragment. A heterozygous sample after denaturation, renaturation of PCR, contains four duplexes:two homoduplexes and two heteroduplexes. After PCR, carried out in the presence of a suitable dye, the product is heated while the level of fluorescence is measured. As the temperature rises and the duplex passes through its melting transition, dye is released and fluorescence intensity is reduced. A single base change can be reflected in the melting temperature difference, the performance difference in the melting curve. High-resolution melting curve analysis had been proved to be a low-cost, high-throughput, rapid, and highly sensitive method for detecting gene mutations.In this study, we mainly through site-directed mutagenesis and gene cloning techniques to construct the known mutations of TMPRSS6 gene, and the exons and intron-exon junctions of TMPRSS6 gene, primers were designed to establish high-resolution melting curve analysis assay. And use the detection method to screening iron deficiency anemia patients in order to find related data of TMPRSS6 gene in the Chinese population.We intend to provide references for further clarify the molecular mechanism of the iron-refractory iron deficiency anemia, and the clinical diagnosis, prevention and treatment of such diseases. And we intend to provide a reference for the molecular mechanism reseach in other abnormal iron metabolism diseases caused by TMPRSS6 gene mutations.Samples and Methods1. SamplesA total of 145 Chinese patients (41 male and 104 female,18.3±14.9 years old) with IDA were recruited from Zhuhai Municipal Maternal and Child Healthcare Hospital (PR China) for the study. Genomic DNA was extracted from 2 ml EDTA-anticoagulated peripheral blood by the standard phenol/chloroform method.2. Hematological analysisBlood routine, hemoglobin content, iron parameters were analyzed in the Zhuhai Institute of Medical Genetics, Zhuhai Municipal Maternal and Child Healthcare Hospital. Detected hepcidin levels with "human hepcidin ELIS A" kit.3. Molecular analysisIn this study, we developed a PCR/HRM assay to detect TMPRSS6 gene mutations. We used site-directed mutagenesis to construct the clone libraries containing the 17 known mutations of TMPRSS6 gene. 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 pTA2-T vector with gene engineering techniques. The host bacterial strain is E. coli DH5a. Each of inserted DNA fragment in these recombinants was sequenced to verify the DNA sequence of mutants cloned.The exons and intron-exon junctions of TMPRSS6 gene, primers were designed to establish HRM analysis assay. All 18 exons and intron-exon junctions of TMPRSS6 gene were amplified in 23 reactions.4. Statistical analysisThe TMPRSS6 gene high-resolution melting assay detected 17 known mutations of TMPRSS6 gene repeatedly. The reproducibility of HRM analysis for detection of the 17 known mutations of TMPRSS6 gene was assessed by examination of the melting temperatures and coefficients of variation. Ten samples for each TMPRSS6 gene known mutation were detected with the HRM assay in triplicate. Statistical analysis was conducted with SPSS 13.0 software.5. TMPRSS6 gene mutation screeningThe collected genomics DNA samples used as templates for PCR amplification, the PCR products subjected to HRM analysis.6. Comprehensive analysis of the experimental results. ResultsUsing megaprimer PCR site-directed mutagenesis method constructed TMPRSS6 gene known mutations, and the mutation fragments were cloned into pTA 2-T vector, and sequencing. All clones were identified as desired mutation, and the achievement ratio of mutagenesis reached 100%.The high-resolution melting curve analysis assay for TMPRSS6 gene can rapidly and accurately distinguish the sequence variation of known mutations and wild-type sequence.We collected 145 samples with iron deficiency anemia, selected 26 samples with high levels of hepcidin. None of the 17 known TMPRSS6 gene mutations was found in any patient, only detected 3 known single nucleotide polymorphisms.DisscussionMicronutrient deficiencies have a high proportion in the malnutrition. Iron deficiency is one of the most popular nutritional diseases all over the world, and iron deficiency anemia is the highest incidence of nutritional diseases of the world. People pay more and more attentions to the diseases caused by iron imbalance in the body.High-resolution melting analysis is a recently developed method for mutation scanning. It is not limited by the type and the site of mutation, sequence-specific probe is unnecessary, and run directly to detect genotype of sample after PCR amplification. Because of its simple, quick, low cost, accurate and the closed-tube operation, high-resolution melting analysis method was widespread concerned.This high-resolution melting assay is a reliable, rapid, and highly sensitive method in distinguishing the known mutations of TMPRSS6 gene from wild-type.This method based on PCR/HRM is a high-throughput, high sensitivity, automation, rapid, accurate and economical mutation detection method.In recent years, there were a set of reports that iron-refractory iron deficiency anemia caused by transmembrane serine protease 6 gene mutations. But there were not reports in TMPRSS6 gene mutation screening. In this study, some patients with IDA were screened with this assay and no TMPRSS6 gene causative mutation was found in any patient. At present, iron intake insufficiently and iron loss excessively is mainly conceived as the etiology of iron deficiency anemia, and the analysis of genetic causes is few. According to the search results in the literature search databases such as PubMed, no related TMPRSS6 gene mutations were reported in Chinese population. Therefore, there is great significance and value in the research of genetic causes in iron deficiency anemia. This study established a low-cost, high-throughput, rapid, and highly sensitive assay for detecting TMPRSS6 gene mutations, which provides an alternative method in gene diagnosis and prenatal diagnosis for a class of diseases caused by TMPRSS6 gene mutations.This study provided important references, including further clarify the molecular mechanism of the iron-refractory iron deficiency anemia, for the clinical diagnosis, prevention and treatment of such diseases. And it provided an important reference for the molecular mechanism reseach in other abnormal iron metabolism diseases caused by TMPRSS6 gene mutations.