Screening Of Gene Mutations In Hereditary Disease By HRM And Detecting β- Thalassemia In Clinic

α-thalassemia,β-thalassemia and DMD are the most common hereditary disease, they severely affect human healthy. At present, only common known mutations and deletion mutations can be detected in clinical, and the point mutation would be miss. For example, DMD gene with 79 exons and several promoters, the point mutation could be present every of them. If the patient caused by point mutation, they do not have the change of diagnosis and their family can not be carried out prenatal diagnosis. Besides,if the mutation ofβ-thalassemia is not common, it could result in the born of severe case. Gene sequencing is the gold standard of detecting point mutations, but it is very expensive and tedious, so, there is emergency request a simple and high sensitive method to detect point mutations. Now, many effects have focus on screening of point mutations first, and them sequencing in order to save time and money. Recently there are many papers report that HRM analysis is a very reliable and simple method to screening of point mutations.PartⅠEstablishing of HRM Analysis to Rapidly Identify of Gene Mutations in Hereditary DiseaseObjectiveEstablishing of HRM analysis to detect gene mutations in hereditary disease.Using HRM analysis assay to rapidly identify of HBB gene,the fourth exon of the DMD gene mutations and mutations ofα-thalassemia(CS,QS and WS). Methods1. First, we used LightScanner96 HRM and two primers, and then, we used LightScanner32 HRM and five primers to detect the most part of the HBB gene respectively. Establishing of the optimal condition for HRM analysis (including the concentration of primers, annealing temperature, concentration of magnesium and so on).2. Seventy six(including twelve homozygous mutations)and one DMD point mutation samples were analyzed with the instrument of LightScanner 32 and LC Green Plus. For the homozygous mutations, after the first detection, we added some wild type samples in the homozygous mutational samples for the second detection.3. We used the metod of scanning and unlabeled oligonucleotide probes to detect mutations ofα-thalassemia(CS, QS and WS).4. Sequencing of the humanβ-globin gene for the samples which were doubted with mutations by ABI 3100.ResultsThe detection of HRM must use the same concentration of DNA; the best TM would elevate about 5℃after adding LC Green Plus. It could be used to identify the commonest of the HBB gene mutations in China, besides, this method can screening of some non-common mutations and homozygous mutations ofβ-thalassemia and DMD. But we fail to detect mutations ofα-thalassemia(CS, QS and WS)with the metod of scanning and unlabeled oligonucleotide probes. ConclusionHRM analysis is a rapid and reliable technique for the identification of gene mutations.Part II Detectingβ- thalassemia in ClinicChapterⅠThe effect of HbA2 on screening ofβ- thalassemiaObjectiveTo evaluate elevated HbA2 (≥3.5%) as an important marker forβ-Thalassemia trait.MethodsHemoglobin capillary electrophoresis was performed on a high performance liquid chromatography (HPLC) using the VARIANT? HPLC system and complete blood count was detected by automated analyzer according to the manufacturer's instructions. The subjects were divided into two groups. The first group who with HbA2 level≥3.5% were detected by the PCR-reverse dot-blot (RDB) assay or direct sequencing of the humanβ-globin gene. The second group had no iron deficiency or microcytic anemia with HbA2 level < 3.5%, and were excludedβ-thalassemia mutation based on noβ-thalassemia history, normal complete blood count and RDB assay. For samples with HbA2 level between 3.0% and 3.5%, and none of the 17 knownβ-thalassemia mutation detected by RDB assay, direct sequencing of the humanβ-globin gene was performed to make sure there were noβ-thalassemia mutations, other samples were detected by RDB assay. The data were subjected to statistical analysis using SPSS 16.0 software. Student's t-test was applied and the P-value calculated,a P value <0.05 was considered significant.Results1. For the first group of the subjects (n = 150, 20-40 years old) that included 65 pregnant women and 85 non-pregnant women with elevated HbA2 level (≥3.5%), RDB assay showed that 147 hadβ-thalassemia mutations and 3 had none of the 17 knownβ-thalassemia mutations. Sequencing analysis of the humanβ-globin gene on these 3 patients and found that two of them had Hb Kaohsiung (c.341T > A) heterozygotic mutation and the others is -90 (C>T)(HBB:c.-140C>T)heterozygotic mutation.2. In the second group (n = 210, 20-40 years old) that included 96 pregnant and 114 non-pregnant women hadβ-thalassemia mutation.3. Comparion of the HbA2 level between 96 pregnant women and 114 non-pregnant women who had noβTT demonstrated that HbA2 level was slightly elevated in the pregnant women (P < 0.05), but it was under 3.5% whether they were pregnant or not.ConclusionHbA2 level≥3.5% is reliable for the screening and diagnosis ofβTT, not affected by the pregnancy condition in South China population.ChapterⅡDetecting HBB Gene Mutations by HRM in ClinicObjectiveRapid genotyping and screening of HBB gene mutations by HRM analysis.Methods1. Detecting some unknown genotype samples with the optimal conditions by HRM analysis.2. Randomly choose sixty unknown samples (including five samples were diagnosed by microcytic anemia and HbA2≥3.5% but no abnormal by RDB assay), detected by HRM analysis assay, and confirmed by RDB or sequencing methods.3. When detecting the samples, add some known mutation samples as positive and negative control.ResultsHRM analysis assay can identified the same mutation with positive control, and we found HBB:c.68A>C,HBB:c.113G>A,HBB:c.319C>G and HBB:c.341T>A mutations which could not be detected by RDB assay, the result is accord with RDB or sequencing methods.ConclusionHRM analysis assay can be used to detected HBB gene mutations.