title: Genetic diagnosis of hypertrophic cardiomyopathy using mass spectrometry DNA arrays and high resolution melting.
abstract: BACKGROUND
  Hypertrophic cardiomyopathy (HCM), a complex myocardial disorder with an autosomal dominant genetic pattern and prevalence of 1:500, is the most frequent cause of sudden death in apparently healthy young people. The benefits of gene-based diagnosis of HCME for both basic research and clinical medicine are limited by the considerable costs of current genetic testing due to the large number of genes and mutations involved in this pathology. However, coupling two high-throughput techniques--mass spectrometry genotyping (MSG) and high resolution melting (HRM)--is an encouraging new strategy for HCM diagnosis. Our aim was to evaluate the diagnostic efficacy of both techniques in this pathology by studying 13 individuals with a clinical phenotype of HCM.
  
  
  METHODS
  Peripheral blood samples were collected from: (i) seven subjects with a clinical diagnosis of HCM, all bearing known mutations previously identified by dideoxy sequencing and thus being used as blinded samples (sample type 1); (ii) one individual with a clinical diagnosis of HCM negative for mutations after dideoxy sequencing of the five most common HCM genes, MYH7, MYBPC3, TNNI3, TNNT2 and MYL2 (sample type 2); and (iii) five individuals individual with a clinical diagnosis of HCM who had not previously been genetically studied (sample type 3).
  
  
  RESULTS
  The 13 samples were analyzed by MSG for 534 known mutations in 32 genes associated with HCM phenotypes and for all coding regions and exon-intron boundaries of the same HCM genes by HRM. The 32 studied genes include the most frequent HCM-associated sarcomere genes, as well as 27 genes with lower reported HCM phenotype association. This coupled genotyping strategy enabled us to identify a c.128delC (p.A43Vfs165) frame-shift mutation in the CSRP3 gene, a gene not usually studied in current HCM genetics. The heterozygous CSRP3 mutation was found in two patients (sample types 2 and 3) aged 50 and 52 years, respectively, both with diffuse left ventricular hypertrophy. Furthermore, this coupled strategy enabled us to find a novel mutation, c.817C >T (p.Arg273Cys), in MYBPC3 in an individual from sample type 3, subsequently confirmed by dideoxy sequencing. This novel mutation in MYBPC3, not present in 200 chromosomes from 200 healthy individuals, affects a codon known to harbor an HCM-causing mutation--p.Arg253His.
  
  
  CONCLUSIONS
  In conclusion, in the cohort used in this work coupling two technologies, MSG and HRM, with high sensitivity and low false positive results, enabled rapid, innovative and low-cost genotyping of HCM patients, which may in the short-term be suitable for accurate genetic diagnosis of HCM.
authors.affiliation: Centro de Metabolismo e Endocrinologia, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Susana
authors.initials: S
authors.lastname: Santos
authors.forename: Vasco
authors.initials: V
authors.lastname: Lança
authors.forename: Helena
authors.initials: H
authors.lastname: Oliveira
authors.forename: Patrícia
authors.initials: P
authors.lastname: Branco
authors.forename: Leonor
authors.initials: L
authors.lastname: Silveira
authors.forename: Vanda
authors.initials: V
authors.lastname: Marques
authors.forename: Dulce
authors.initials: D
authors.lastname: Brito
authors.forename: Hugo
authors.initials: H
authors.lastname: Madeira
authors.forename: Manuel
authors.initials: M
authors.lastname: Bicho
authors.forename: Alexandra R
authors.initials: AR
authors.lastname: Fernandes
mesh_terms: D000328:Adult; D024722:Cardiac Myosins / Q000235:genetics; D002312:Cardiomyopathy, Hypertrophic / Q000150:complications / Q000175:diagnosis* / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics; D016022:Case-Control Studies; D016757:Death, Sudden, Cardiac / Q000209:etiology; D005260:Female; D005820:Genetic Testing; D006801:Humans; D020930:MAP Kinase Kinase Kinases / Q000235:genetics; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D009154:Mutation / Q000235:genetics*; D018995:Myosin Heavy Chains / Q000235:genetics; D018994:Myosin Light Chains / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010641:Phenotype; D017346:Protein Serine-Threonine Kinases; D020107:Troponin T / Q000235:genetics; D000099163:Myosin Binding Protein C
chemical_list: D024722:Cardiac Myosins; D002352:Carrier Proteins; D020930:MAP Kinase Kinase Kinases; D018995:Myosin Heavy Chains; D018994:Myosin Light Chains; D017346:Protein Serine-Threonine Kinases; D020107:Troponin T; D000099163:Myosin Binding Protein C; C466107:MRLC2 protein, human; C501641:MYH7 protein, human; C545568:TNNT2 protein, human; C481078:TNNI3K protein, human