Detection rates and phenotypic spectrum of m.3243A > G in the MT-TL1 gene: A molecular diagnostic laboratory perspective
Introduction
The disorder characterised by Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS) is a well-recognised syndrome in the field of mitochondrial diseases. Goto and colleagues (Goto et al., 1990) were among the first to identify the most common point mutation associated with MELAS m.3243A > G, located in the tRNA leucine (UUR) gene (MT-TL1) of the mitochondrial genome. In their study, genetic testing on skeletal muscle biopsies of patients with MELAS revealed 80% of these patients harboured the point mutation (Goto et al., 1990, Goto et al., 1991). The consequent invariant criteria established for the clinical diagnosis of MELAS includes (1) stroke-like episodes before the age of 40 years; (2) encephalopathy characterized by seizures, dementia, or both; and (3) lactic acidosis, ragged-red fibres (RRF) or both. Diagnosis would be considered certain if there are also at least two of the following: normal early development, recurrent headache or recurrent vomiting (Ciafaloni et al., 1992, Hirano et al., 1992, Pavlakis et al., 1984).
Subsequent to the seminal reports, the clinical spectrum of the m.3243A > G mutation has been recognised to be much more diverse and very heterogeneous (Ciafaloni et al., 1992, Jean-Francois et al., 1994, Nesbitt et al., 2013) and there are many other distinct syndromes reported in association with MT-TL1 m.3243A > G, such as Myoclonic Epilepsy and Ragged Red Fibres (MERRF) (Fabrizi et al., 1996), MERRF/MELAS overlap syndrome (Campos et al., 1996), Progressive External Ophthalmoplegia (PEO) (Moraes et al., 1993), Chronic Progressive External Ophthalmoplegia (CPEO) (Bosbach et al., 2003, Koga et al., 2000, Mariotti et al., 1995), MERRF/CPEO overlap syndrome (Verma et al., 1996), Maternally Inherited Diabetes and Deafness (MIDD) (Van den Ouweland et al., 1992) and Leigh’s syndrome (Koga et al., 2000). Non-syndromic phenotypes associated with this point mutation include hypertrophic cardiomyopathy (Koga et al., 2000, Silvestri et al., 1997), cluster headache (Shimomura et al., 1994), pancreatitis (Kishnani et al., 1996) subacute dementia with myoclonus mimicking Creutzfeldt-Jakob Disease (Isozumi et al., 1994) and myoclonous with ragged red fibre phenotype (Nesbitt et al., 2013). According to the studies by Chinnery et al. (1999) and Frederiksen et al. (2006), there appears to be a uniform distribution of mutant mtDNA throughout the three germ layers in embryogenesis; however, there are significant differences between heteroplasmic levels of the individual tissue types, indicating tissue-specific segregation of MT-TL1 m.3243A > G later in embryogenesis, which is believed to explain the diversity of clinical phenotypes.
Therefore, a series of ancillary clinical and laboratory investigations including biochemical assays (lactic acid levels and respiratory chain complex activities), neuroimaging (computed tomographic (CT) or magnetic resonance imaging (MRI) for evidence of focal brain abnormalities) (Sparaco et al., 2003), tissue histochemistry (to detect the presence of ragged red fibres or cytochrome-oxidase deficient fibres), and molecular mitochondrial DNA analysis need to be considered in order to provide a definitive diagnosis for MELAS syndrome.
In this report, we describe the detection rate of the MT-TL1 m.3243A > G mutation and the associated clinical features for 745 adult patients referred for mitochondrial genetic screening specifically directed at this mutation in the setting of a suspected mitochondrial disorder. A detailed summary of the associated clinical features is discussed, including evaluation of the percentage of patients fulfilling the invariant criteria for MELAS as compared to those who manifest only some of the symptoms or show other distinct clinical features associated with this point mutation.
Section snippets
Patients and methods
From 2002 to 2012, our laboratory received specimens from 1673 patients with a suspected mitochondrial disorder, of which 745 cases were screened for the MT-TL1 m.3243A > G mutation. The majority of referrals were from Victoria with some from New South Wales and Tasmania. Requisition forms are routinely sent out to referring medical practitioners, with the majority being neurologists, to accompany the patient specimens in order to obtain informed genetic consent and to indicate the salient
Clinical features of patients with MT-TL1 m.3243A > G mutation
Over the period of 11 years, a total of 745 patients were systematically screened for MT-TL1 m.3243A > G with 187 cases solely screened for this specific mutation. Sequencing of the entire MT-TL1 gene was carried out in 160 patients to search for other uncommon sequence variants, including m.3252A > G, m.3256C > T and m.3291 T > C associated with MELAS. Of the 160 patients referred from 2010 to 2012 that had their entire MT-TL1 gene screened, only the common m.3243A > G point mutation was detected in eight
Heterogeneous clinical spectrum of MT-TL1 m.3243A > G
The seminal studies by Goto and colleagues reported the point mutation MT-TL1 m.3243A > G to be associated with approximately 80% of well-defined MELAS cases (Goto et al., 1990, Goto et al., 1991). Similarly in another study by Ciafaloni et al. (1992), they screened 23 patients (from 21 unrelated pedigrees) who fulfilled the reported invariant clinical criteria for MELAS, as well as 25 oligo-symptomatic and asymptomatic relatives and 50 disease controls with documented or suspected mitochondrial
Conclusion
We reviewed our mitochondrial genetic screening over a period of 11 years and observed relatively low mutation detection rates for MT-TL1 m.3243A > G, with the majority of patients found to harbour the MT-TL1 m.3243A > G mutation not fulfilling the invariant diagnostic criteria for MELAS. Indeed, most patients were oligo-symptomatic or mono-symptomatic with only one or more of the invariant criteria (without stroke) with maternal inheritance or family history. Importantly, mono-symptomatic patients
Funding
St. Vincent’s Melbourne Neuromuscular Diagnostic Laboratory is funded by the Department of Human Services, Victoria, Australia.
Acknowledgements
Centre of Translational Pathology, Department of Pathology, Melbourne University and Victorian Neuromuscular Diagnostic Laboratory, Alfred Hospital.
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These two authors contributed equally to this work.