The Application Of High-resolution Melting Analysis In Diagnosis And Prenatal Diagnosis Of Single-gene Genetic Diseases

Alpha-thalassemia is one of the most commonly inherited single-gene disorders in southern China. This disorder usually results from deletions or point mutations ofα-globin genes. The severest form ofα-thalassemia compatible with postnatal life is hemoglobin H (Hb H) disease. Two commonly molecular types of it are deletional Hb H disease and nondeletional Hb H disease. Patients with nondeletional Hb H disease have more severe anemia, hemolysis and splenomegaly, and are more likely to require transfusions than those with deletional Hb H disease. It is significant to use a molecular method to screenα-globin genes variants for detecting carriers and prenatal diagnosing the major Hb H disease. In Chinese individuals the two common nondeletional variants are Hb Constant Spring and Hb Quong Sze. Hb Constant Spring can be detected by capillary electrophoresis (CE)or high performance liquid chromatography (HPLC), whereas Hb Quong Sze can't be detected by routine screening methods for it is highly unstable. In this study, we explored the feasibility of high-resolution melting analysis (HRM) for molecular identification of Hb Quong Sze.ObjectionTo explore the feasibility of using high-resolution melting curves to detect Hb Quong Sze.Methods1. During 2007 to 2010, a total of 58 DNA samples were selected from prenatal diagnostic center of Guangzhou maternal and neonatal hospital, including 11 Hb QS(α^QS α/αα),9 Hb H-QS (α^QSα/--^SEA),1 Hb QS-α^4.2(α^QSα/-α^4.2) and 37 normal controls . All samples were genotyped by gap-PCR, reverse dot blotting and DNA sequencing.2. DNA sequence ofα-globin genes were obtained from UCSC Genome Bioinformatics (http://genome.ucsc.edu/). Using LightScanner Primer Design Software, primers were designed to amplify simultaneously bothα1- andα2-globin genes covering the loci of the c.377 (codon 125) mutation.3. Optimized PCR reaction and successfully amplified specific target fragment.4. After PCR amplification, the PCR plates were transferred to a 96-well LightScanner .The fluorescence data were collected from 70℃to 96℃for amplicon scanning. Melting curves were analyzed by LightScanner Software Call-IT 2.0 with normalized, temperature-shifted curve and difference plots.Results1. All samples were successfully amplified and obtained good HRM melting curve.2. All the Hb Quong Sze mutations were successfully differentiated from the wild type controls by high-resolution melting curve analysis. 11 Hb QS(α^QSα/αα) integrated one cluster,9 Hb QS-H(α^QSα/--^SEA) and 1 Hb QS-α^4.2(α^QSα/-α^4.2) integrated another cluster and 37 normal controls integrated the third cluster.ConclusionHRM is a rapid, cheap and closed-tube method with high sensitivity and specificity in scanning for Hb Quong Sze. Fetal short-limb dysplasias are a group of skeletal dysplastic diseases with shortened long bones in common. Most of them are constitutional bone diseases, excluding some chromosomal diseases.There are two type of it: lethal and nonlethal skeletal dysplasia. Thanatophoric dysplasia is the common lethal dysplasia. It is usually aporadic and probably results from a new autosomal dominant mutation. The prevalence of it ranges from 0.21 to 0.30 per 10 000 births.Thanatophoric dysplasia is divided in two types. TD I is most common and is characterized by short, curved tubular bones, and short ribs with platyspondyly. The diagnosis of TD I mainly depend on sonagraphic scanning. Sonagraphic technology is valuable in the prenatal diagnosis of some TD I cases. However, because another TD I cases just show short-limb which can be found in other short-limb dysplasias and lack of specific sonographic signs, it is really difficult for sonagraphy to attribute a skeletal dysplasia to a detailed type. The genetic diagnosis is the best criteria for identifying TD I. TD I is related to the mutation of fibroblast growth factor receptor 3 (FGFR3). The p.R248C,p.S249C and p.Y373C mutations of FGFR3 lesds to 73% TD I. In this study, we try to establish a rapid molecular method for diagnosis TD I by using high-resolution melting analysis to scan the C742T mutation in exon 7 of FGFR3.Objective1. To establish a rapid molecular method for diagnosis type 1 thanatophoric dysplasia.2. To explore the feasibility of using high-resolution melting curve analysis to detect the c.742C > T (p.R248C) mutation of the FGFR3 gene causing type 1 thanatophoric dysplasia. Methods1. DNA samples, including 10 prenatal samples with type 1 TD harboring the c. 742 C > T variant identified previously by DNA sequencing and 30 control samples from healthy individuals, were selected from prenatal diagnostic center of Guangzhou maternal and neonatal hospital.2. DNA sequence of FGFR3 gene was obtained from UCSC Genome Bioinformatics (http://genome.ucsc.edu/). Using LightScanner Primer Design Software, primers were designed to amplify FGFR3 genes covering the loci of the c.742 (codon 248) mutation.3. Optimized PCR reaction and successfully amplified specific target fragment.4. After PCR amplification, the PCR plates were transferred to a 96-well LightScanner .The fluorescence data were collected from 70℃to 98℃for amplicon scanning. Melting curves were analyzed by LightScanner Software Call-IT 2.0 with normalized, temperature-shifted curve and difference plots.ResultsUsing the high-resolution melting curve analysis, we successfully genotyped the c. 742C > T (p. R248C) mutation in all of the 10 tested samples with type 1 TD.ConclusionHigh-resolution melting analysis is a kind of easy-to-use ,rapid , and sensitive one tube assay for genotyping the c. 742C > T (p. R248C) mutation which is the most common one associated with type 1 TD. The technology has the potential to reduce sequencing burden and would be suitable for mutations screening of FGFR3 gene that have been found to be associated with TD.