Mitochondrial cardiomyopathies: how to identify candidate pathogenic mutations by mitochondrial DNA sequencing, MITOMASTER and phylogeny

Abstract

Pathogenic mitochondrial DNA (mtDNA) mutations leading to mitochondrial dysfunction can cause cardiomyopathy and heart failure. Owing to a high mutation rate, mtDNA defects may occur at any nucleotide in its 16 569 bp sequence. Complete mtDNA sequencing may detect pathogenic mutations, which can be difficult to interpret because of normal ethnic/geographic-associated haplogroup variation. Our goal is to show how to identify candidate mtDNA mutations by sorting out polymorphisms using readily available online tools. The purpose of this approach is to help investigators in prioritizing mtDNA variants for functional analysis to establish pathogenicity. We analyzed complete mtDNA sequences from 29 Italian patients with mitochondrial cardiomyopathy or suspected disease. Using MITOMASTER and PhyloTree, we characterized 593 substitution variants by haplogroup and allele frequencies to identify all novel, non-haplogroup-associated variants. MITOMASTER permitted determination of each variant's location, amino acid change and evolutionary conservation. We found that 98% of variants were common or rare, haplogroup-associated variants, and thus unlikely to be primary cause in 80% of cases. Six variants were novel, non-haplogroup variants and thus possible contributors to disease etiology. Two with the greatest pathogenic potential were heteroplasmic, nonsynonymous variants: m.15132T>C in MT-CYB for a patient with hypertrophic dilated cardiomyopathy and m.6570G>T in MT-CO1 for a patient with myopathy. In summary, we have used our automated information system, MITOMASTER, to make a preliminary distinction between normal mtDNA variation and pathogenic mutations in patient samples; this fast and easy approach allowed us to select the variants for traditional analysis to establish pathogenicity.

Figure 1

Six novel, non-haplogroup variants as potential pathogenic mtDNA mutations. Shown are chromatograms for six potential mtDNA mutations m.15132T>C, MT-CYB (p.M129T) in case 2; m.15324C>T, MT-CYB (p.A193V) in case 3; m.11069A>G, MT-ND4 (p.I104V) in case4; m.15222A>G, MT-CYB (p.D159G) in NonCM1; m.8954T>C in MT-ATP6 (p.I143T) in NonCM6; m.6570G>T, MT-CO1 (p.A223S) in NonCM8. Heteroplasmy was detected in three, that is, case 2, NonCM1 and NonCM8.

Figure 2

Pedigree and clinical features for two cases with novel, potential mtDNA mutations: case 2 (m.15132T>C, MT-CYB) and NonCM8 (m.6570G>T, MT-CO1). Pedigree analysis and clinical screening by echocardiography suggested (case 2) or supported (NonCM8) matrilineal inheritance of mitochondrial disease. Circles represent females and squares represent males; solid shapes are affected individuals; an arrow indicates the proband. Number is the age in years at last clinical evaluation or age of death. (a) Case 2: dilated cardiomyopathy (DCM). Family history included maternal relatives with early stroke (<55 years), diabetes and questionable cardiomyopathy. (b) NonCM8: myopathy. Family history included the mother with myopathy and maternal grandmother with diabetes. Abbreviations: AD, Alzheimer's disease; BL HTN, borderline hypertension; ca, cancer; d., died; CMP (?), questionable diagnosis of cardiomyopathy by history; ECG nl, electrocardiogram results normal; ECHO nl, echocardiography results normal; HT, hypertriglyceridemia; LBBB, left bundle branch block; NIDDM, non-insulin dependent diabetes mellitus; NSVT, nonsustained supraventricular tachycardia; PFO, patent foramen ovale.

Figure 3

Approach to identify potential mtDNA mutations. Four major steps to facilitate the selection of potential mtDNA mutations. ^*Rare variants require additional analysis before exclusion.