High resolution melting: improvements in the genetic diagnosis of hypertrophic cardiomyopathy in a Portuguese cohort

Abstract

Background: Hypertrophic Cardiomyopathy (HCM) is a complex myocardial disorder with a recognized genetic heterogeneity. The elevated number of genes and mutations involved in HCM limits a gene-based diagnosis that should be considered of most importance for basic research and clinical medicine.

Methodology: In this report, we evaluated High Resolution Melting (HRM) robustness, regarding HCM genetic testing, by means of analyzing 28 HCM-associated genes, including the most frequent 4 HCM-associated sarcomere genes, as well as 24 genes with lower reported HCM-phenotype association. We analyzed 80 Portuguese individuals with clinical phenotype of HCM allowing simultaneously a better characterization of this disease in the Portuguese population.

Results: HRM technology allowed us to identify 60 mutated alleles in 72 HCM patients: 49 missense mutations, 3 nonsense mutations, one 1-bp deletion, one 5-bp deletion, one in frame 3-bp deletion, one insertion/deletion, 3 splice mutations, one 5'UTR mutation in MYH7, MYBPC3, TNNT2, TNNI3, CSRP3, MYH6 and MYL2 genes. Significantly 22 are novel gene mutations.

Conclusions: HRM was proven to be a technique with high sensitivity and a low false positive ratio allowing a rapid, innovative and low cost genotyping of HCM. In a short return, HRM as a gene scanning technique could be a cost-effective gene-based diagnosis for an accurate HCM genetic diagnosis and hopefully providing new insights into genotype/phenotype correlations.

Figure 1

a) Melting curves of exon 19 of the MYBPC3 gene (NM_000256). b) Difference plot of the melting curves of exon 19 of the MYBPC3 gene. The arrows in both figures indicate the wild-type (wt) profile and patient 16 respective variations. Two healthy control individuals were used has a reference curve. c) Chromatogram analysis resulting from sequencing exon 19 of the MYBPC3 gene in patient 16 with the c.1945insT, 1941-1946delCCTGGA, p.Pro647fs. This mutation was also present in patient 17. d) Melting curves of exon 22(1) of the MYH7 gene (NM_000257). e) Difference plot of the melting curves of exon 22(1) of the MYH7 gene. The arrows in both figures indicate the wild-type (wt) profile and patient 13 respective variations. Five healthy control individuals were used has a reference curve. f) Chromatogram analysis resulting from sequencing exon 22 of the MYH7 gene in patient 13. g) Polyphen analysis of p.Ala862Val genetic variation http://genetics.bwh.harvard.edu/pph2/. h) Conservation across species of codon 862 of the β-myosin protein (source: http://www.uniprot.org/). The box shows the amino acid changed by the MYH7 c.2585 C > T mutation. [BOVIN - Bos Taurus; HUMAN - Homo sapiens; MOUSE - Mus musculus; RAT - Rattus norvegicus; PIG - Sus scrofa].

Figure 2

a) Melting curves of exon 2 of the MYH6 gene (NM_002471). b) Difference plot of the melting curves. The arrows in both figures indicate the wild-type (wt) profile and the patient respective variation. Seven healthy control individuals were used has a reference curve. c) Chromatogram analysis resulting from sequencing exon 2 of the MYH6 gene in patient 37. d) Polyphen analysis of p.Gly56Arg genetic variation http://genetics.bwh.harvard.edu/pph2/. e) Conservation across species of codon 56 of the α-myosin light chain protein (source: http://www.uniprot.org/). The box shows the amino acid changed by the MYH6 c.220 G > A mutation. [HUMAN - Homo sapiens; MOUSE - Mus musculus; RAT - Rattus norvegicus; DANRE - Danio rerio].

Figure 3

a) Melting curves of exon 14 of the TNNT2 gene (NM_000364). b) Difference plot of the melting curves. The arrows in both figures indicate the wild-type (wt) profile and the patient respective variation. Eight healthy control individuals were used has a reference curve. c) Chromatogram analysis resulting from sequencing exon 14 of the TNNT2 gene in patient 22. d) Polyphen analysis of p.Lys241Met genetic variation http://genetics.bwh.harvard.edu/pph2/. e) Conservation across species of codon 241 of the Troponin T protein (source: http://www.uniprot.org/). The box shows the amino acid changed by the TNNT2 c.722A > T mutation. [HUMAN - Homo sapiens; MOUSE - Mus musculus; RAT - Rattus norvegicus; BOVIN - Bos Taurus; DANRE - Danio rerio].

Figure 4

a) Melting curves of exon 3 of the MYL2 gene (NM_000432). b) Difference plot of the melting curves. The arrows in both figures indicate the wild-type (wt) profile and the patient respective variation. Eight healthy control individuals were used has a reference curve. c) Chromatogram analysis resulting from sequencing exon 3 of the MYL2 gene in patient 35. d) Polyphen analysis of p.Ile44Met genetic variation http://genetics.bwh.harvard.edu/pph2/. e) Conservation across species of codon 44 of the regulatory myosin light chain protein (source: http://www.uniprot.org/). The box shows the amino acid changed by the MYL2 c.132 T > G mutation. [HUMAN - Homo sapiens; MOUSE - Mus musculus; RAT - Rattus norvegicus; BOVIN - Bos Taurus; RABIT - Oryctolagus cunic].

Figure 5

a) Melting curves of exon 14 of the TNNT2 gene (NM_000364). b) Difference plot of the melting curves. The arrows in both figures indicate the wild-type (wt) profile and the patients respective variations. Nine healthy control individuals were used has a reference curve. c) Chromatogram analysis resulting from sequencing exon 14 of the TNNT2 gene in patient 67. d) Polyphen analysis of p.Lys260Arg genetic variation http://genetics.bwh.harvard.edu/pph2/. e) Conservation across species of codon 260 of the cardiac Troponic T protein (source: http://www.uniprot.org/). The box shows the amino acid changed by the TNNT2 c.778A > G mutation. [HUMAN - Homo sapiens; MOUSE - Mus musculus; RAT - Rattus norvegicus; CANFA - Canis familiaris; RABIT - Oryctolagus cunic]. f) Melting curves of the population study of exon 14 of the TNNT2 gene (NM_000364). g) Difference plot of the melting curves. The arrows in both figures indicate the wild-type (wt) profile and the c.778A > G (p.Lys260Arg) variation on two chromosomes of control individuals. 100 healthy control individuals (200 chromosomes) were used has a reference curve.