Searching for a needle in the haystack: Comparing six methods to evaluate heteroplasmy in difficult sequence context Ivana Kurelac a , Martin Lang a , Roberta Zuntini a , Claudia Calabrese a , Domenico Simone b , Saverio Vicario c , Monica Santamaria c , Marcella Attimonelli b , Giovanni Romeo a, ⁎, Giuseppe Gasparre a a Dip. Scienze Ginecologiche, Ostetriche e Pediatriche, Genetica Medica, Pol. Universitario S.Orsola-Malpighi, 40138, Bologna, Italy b Dipartimento di Biochimica e Biologia Molecolare “E. Quagliariello”, Università di Bari, 70126, Bari, Italy c Istituto di Tecnologie Biomediche, CNR, 70126, Bari, Italy abstract article info Available online 13 June 2011 Keywords: Mitochondrial DNA mutations Heteroplasmy Indels Homopolymeric stretch Mitochondrial DNA (mtDNA) mutations have been involved in disease, aging and cancer and furthermore exploited for evolutionary and forensic investigation. When investigating mtDNA mutations the peculiar aspects of mitochondrial genetics, such as heteroplasmy and threshold effect, require suitable approaches which must be sensitive enough to detect low-level heteroplasmy and, precise enough to quantify the exact mutational load. In order to establish the optimal approach for the evaluation of heteroplasmy, six methods were experimentally compared for their capacity to reveal and quantify mtDNA variants. Drawbacks and advantages of cloning, Fluorescent PCR (F-PCR), denaturing High Performance Liquid Chromatography (dHPLC), quantitative Real-Time PCR (qRTPCR), High Resolution Melting (HRM) and 454 pyrosequencing were determined. In particular, detection and quantification of a mutation in a difficult sequence context were investigated, through analysis of an insertion in a homopolymeric stretch (m.3571insC). © 2011 Elsevier Inc. All rights reserved. 1. Introduction 1.1. Mitochondrial genetics and questions inherent to heteroplasmy estimates Mitochondrial DNA mutations have been implicated in disease, aging and cancer (Wallace, 1999) and also exploited for forensic (Jarman et al., 2009), phylogenetic and population genetic studies (Torroni et al., 2006). Due to the mitochondrial genome physiological polyploidy, different mtDNA genotypes may coexist within a single cell, a condition known as heteroplasmy. It is generally accepted that a threshold of heteroplasmy must be reached in order for a mtDNA mutation to display a phenotypic effect (Rossignol et al., 2003). In fact, one of the criteria to infer the pathogenicity of a mtDNA change is its level of heteroplasmy (Chinnery and Bindoff, 2003). In the case of Maternally Inherited Diabetes and Deafness (MIDD), Neurogenic muscle weakness, Ataxia and Retinitis Pigmentosa (NARP) and Leigh syndromes, correlation between clinical severity of the disease and the proportion of mutant mtDNA has been demonstrated (Carelli et al., 2002; Laloi-Michelin et al., 2009). Moreover, it has been reported that only heteroplasmic, not homoplasmic, mtDNA muta- tions appear to promote tumorigenesis (Park et al., 2009) and we have recently shown how different loads of disruptive complex I mtDNA mutations produce nearly opposite consequences on the metabolic homeostasis of cancer cells (Porcelli et al., 2010). 1.2. Methods for mtDNA mutation analysis Due to the clinical implications of the threshold effect, which may determine the differential expression of a disease phenotype, a precise quantification of the mtDNA mutant load and efficient detection of low-level heteroplasmies are of paramount importance. Nevertheless, optimization of heteroplasmy evaluation methods has been scarce until now. Restriction-based approaches such as Restriction Fragment Length Polymorphism (RFLP) (Holt et al., 1990), Last Hot Cycle (Moraes et al., 2003) and Fluorescent PCR (F-PCR) (Gigarel et al., 2005), although widely used, may provide inaccurate results due to incomplete digestion and are limited to exclusive recognition of mutations which induce a change in a restriction enzyme consensus sequence (Moraes et al., 1992; Wong and Boles, 2005). Gel-based Biotechnology Advances 30 (2012) 363–371 Abbreviations: mtDNA, mitochondrial DNA; MIDD, Maternally Inherited Diabetes and Deafness; NARP, Neurogenic muscle weakness, Ataxia and Retinitis Pigmentosa; RFLP, Restriction Fragment Length Polymorphism; F-PCR, Fluorescent PCR; TTGE, Temporal Temperature Gradient Gel Electrophoresis; qRT-PCR, quantitative Real-Time PCR; dHPLC, denaturing High Performance Liquid Chromatography; HRM, High Resolution Melting; indels, small insertions and deletions; MT-ND1, mitochondrially encoded NADH dehydrogenase 1; NumtS, Nuclear Mitochondrial Sequences; QS, Quality Score; PGD, Preimplantation Genetic Diagnosis. ⁎ Corresponding author at: Dip Scienze Ginecologiche, Ostetriche e Pediatriche, U.O. Genetica Medica, Policlinico S. Orsola Malpighi, Via Massarenti 9, 40138 Bologna, Italy. Tel.: +39 051 2088 420; fax: +39 051 2088 416. E-mail address: romeo.genetica@yahoo.it (G. Romeo). 0734-9750/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2011.06.001 Contents lists available at ScienceDirect Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv