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Screening of Gene Mutations in Hereditary Disease by HRM and Detecting β- thalassemia in Clinic

Author: OuZhanHui
Tutor: SunZuoFang
School: Guangzhou Medical College
Course: Obstetrics and Gynaecology
Keywords: β- thalassemia Hemoglobin A2 HRM Gene mutation Screening
CLC: R725.5
Type: Master's thesis
Year: 2011
Downloads: 97
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Abstract

α-thalassemia, β-thalassemia and DMD are serious harm to human life and health of the most common single-gene genetic diseases, at present, only for common clinical point mutations and deletions repeat mutations were detected, and because point mutations causative in children or carriers often missed or not genetic diagnosis. If DMD gene, due to its large and no gene mutation hot spots, in clinical repeated only for the deletion and mutation detection, and the point mutation genetic diagnosis of disease patients are deprived of the opportunity, but also means that they can not be DMD families prenatal diagnosis; β-thalassemia mutations if not a common mutation may cause severe birth of children. Sequencing is the gold standard for detection of point mutations but cumbersome and costly workload. Therefore an urgent need to find a simple and highly sensitive mutation detection method, a lot of research has been devoted by first point of the gene mutation screening, screening out suspected serial point mutations after sequencing, thus greatly reducing the cost and workload . Recently a lot of literature that HRM analysis is a good new point mutation detection methods. The first part of the establishment of HRM analysis detection of single gene disorders purpose of point mutations in β-globin, DMD and α-globin gene as the research object, the establishment of a single application of HRM analysis to detect gene point mutation genetic diseases and explore the method apply to homozygous mutation. Method 1 using LightScanner96 and LightScanner32 HRM-analyzer, combined with agarose gel electrophoresis pairs of primers, primer concentration, primer optimal annealing temperature, magnesium ion concentration, the initial template amount and time to explore the dye added. 2 pairs 76 contains both normal and point mutations of the β-globin samples were detected, of which 12 are common homozygous mutant genotype samples homozygous mutations of these specimens are two ways to detect, for the first conventional mutation screening scan and then add an equal proportion of the wild-type samples after mixing scan testing. And this method on 8 DMD, one of which is known DMD exon 4 point mutations were detected. 3 with a mutation screening scanning method and asymmetric PCR combined closed unlabeled probe method 24 with normal and α-thalassemia CS, QS, WS three kinds of point mutations specimens. 4 suspected presence of point mutations in samples with ABI 3100 DNA sequencing instrument for the β-globin gene sequencing. Results HRM detect point mutations, it is necessary to dilute the initial concentration of the template for the same concentration; optimal primer annealing temperature than the temperature before adding the dye to be higher. This method can be used β-globin mutation scanning screening results with RDB method or sequencing methods consistent; For a male patient DMD exon 4 point mutation specimens and twelve CD41/42, IVS-ΙΙ- 654, TATAbos -28, CD17 specimens were homozygous mutations can be scanned directly after mixing and wild-type samples to distinguish between two methods for scanning. The asymmetric PCR combined with non-labeled probe for α-closed thalassemia CS, QS, WS three kinds of point mutations specimens failed, need to be further optimized. Conclusion This study established the application of HRM analysis method for single-point mutation genetic disease detection platform that can quickly and reliably heterozygous and homozygous for mutations of genes were detected. The second part of the β-thalassemia clinical detection of the first chapter HbA2 β-thalassemia screening Objective To evaluate the role of HbA2 elevated (≥ 3.5%) screening β? Thalassemia role. Methods automatic blood analyzer 360 women blood test, hemoglobin electrophoresis HPLC HbA2 level detection. The first group of HbA2 level ≥ 3.5% of 65 pregnant women, 85 were non-pregnant women, underwent RDB or β-globin gene sequencing to detect, and the second group was HbA2 level lt; 3.5% of 96 pregnant women, 114 non-pregnant women, if the MCH gt; 29pg, MCV gt; 90fl is no genetic testing, HbA2 value of 3.0% -3.5% for β-globin gene sequencing, for the remaining detected RDB. Using the t-test statistical value of HbA2. 1 The first group (n = 150 名 ,20-40 years old), of which 65 named pregnant 85 were non-pregnant women, their HbA2 level ≥ 3.5%, RDB detect common display 147 named China β-thalassemia mutations, of which three were normal with RDB detected by sequencing analysis found two cases of RDB can not detect Hb Kaohsiung heterozygous point mutation that HBB: c.341T gt; A heterozygous mutation in the third case of -90 (C gt; T) heterozygous point mutation that HBB: c.-140C gt; T heterozygous point mutations. (2) the second group (n = 210, 20-40 years of age), of which 96 pregnant women, 114 non-pregnant women, RDB method tests showed were not found β-thalassemia mutations and detected by sequencing HbA2 range 3.0% -3.5% of the specimens, also found no presence of β-thalassemia mutations. 3 of pregnant women do not carry thalassemia compared with non-pregnant women, pregnant women, slightly elevated HbA2 levels (P lt; 0.05), but they did not exceed the level of HbA2 or 3.5%. Conclusion Although pregnancy can slightly elevated levels of HbA2, but did not affect the HbA2 for β-thalassemia screening role, both in pregnant and non-pregnant women, HbA2 are very good screening β-thalassemia The value of carriers. Chapter II Application HRM analysis detects β? Thalassemia gene mutation objective application of the above analysis methods established HRM unknown genotype specimens β-globin gene detection to explore the feasibility of its clinical application. Method 1. Apply the conditions of the first part of the optimization method for testing specimens of unknown genotype. 2 Select the 60 specimens, which contains five confirmed hematologic and HbA2 β? Thalassemia RDB method but did not carry abnormal specimens, the first for HRM analysis testing then RDB or β-globin sequencing test for comparing results . 3 for HRM analysis testing to join the Chinese most common β? Thalassemia heterozygous genotype samples for the positive control and the normal wild-type samples as negative controls. Results P1 P2 primers in the reaction product, a sample with the RDB and sequencing results compared to false-positive findings. Other mutations detected in specimens of curves consistent with the positive control genotyping. 5 Tips Hematology outgoing β? Thalassemia specimens were carrying an abnormality is detected during the scan curve detected by sequencing is recognized as an Hb G-Coushatta that HBB: c.68A gt; C, a Cd37 (TGG gt ; TAG) that HBB: c.113G gt; A, a Hb L'Aquila that HBB: c.319C gt; G and two Hb New York that HBB: c.341T gt; A heterozygous mutation, the remaining results with RDB sequencing method or the same method to determine the genotype. Conclusion HRM analysis method can be used clinically β? Thalassemia mutations for rapid genotyping and mutation scanning detection.

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