Mitochondrial respiratory chain diseases (RCDs) are a group of inherited disorders of energy metabolism. Together, they form one of the most common groups of inherited metabolic diseases, with a minimum birth prevalence of 1 of 5000 (Calvo et al., 2006; Mancuso et al., 2007; Neupert and Herrmann, 2007). The diagnosis of mitochondrial RCDs remains a major challenge to the clinician because of the varied clinical presentations, frequent dependence on invasive testing procedures, the huge genetic heterogeneity, and the absence of a reliable screening or diagnostic biomarker that is both sensitive and specific in all cases (Haas et al., 2007; Wong et al., 2010). Diagnostic criteria have been proposed that attempt to take into account clinical manifestations, enzymatic and physiologic analyses, tissue histochemical results, levels of biochemical analytes, and DNA analysis for a more reliable characterization of patients (Nissenkorn et al., 1999; Bernier et al., 2002; Nonaka, 2002; Wolf and Smeitink, 2002; Morava et al., 2006).
Many diseases involve multiple organ systems, in part reflecting the dependence on the energy derived from oxidative phosphorylation in a wide variety of tissues, and this feature may be the initial indication of the correct diagnosis. Any organ or tissue may be involved in this group of diseases (Haas et al., 2007; Wong et al., 2010). There are a number of ‘classic’ clinical syndromes with stereotypical features such as MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), MERRF (myoclonic epilepsy and ragged red fibers), Leigh’s syndrome (LS) (subacute necrotizing encephalomyelopathy), and NARP (neurogenic muscle weakness, ataxia, and retinitis pigmentosa). However, many patients show only nonspecific features of developmental delay or regression, further hindering accurate diagnosis (McFarland and Turnbull, 2009; Wong et al., 2010).
Mitochondrial RCDs have a genetic etiology and this genetic abnormality may be found in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) (Jacobs and Turnbull, 2005; Greaves et al., 2006). Although molecular testing is widely viewed as definitive, confirmation of the diagnosis by molecular methods often remains a challenge because of the large number of genes, the two-genome complexity, and the varying proportions of pathogenic mtDNA molecules in a patient, a concept termed heteroplasmy. Screening of common point mutations and large deletions in mtDNA is typically the first step in the molecular diagnosis (Wong et al., 2010). Although effective treatments remain elusive, a definitive diagnosis is crucial for appropriate symptom management, as well as accurate prognostic and proper genetic counseling (Haas et al., 2007). Thus, identification of the most common mutation in a population facilitates targeting and confirmation of the diagnosis. In this respect, our aim was to choose seven common mutations reported in different populations in order to identify the existing panel of mutations in this group of Egyptian patients with mitochondrial RCDs.
Participants and methods
Participants and investigations
Twenty-eight Egyptian patients from 25 unrelated families with suspected mtDNA disorders were included in this study. Informed consent was obtained from all parents according to the guidelines of the Ethical Committee of the National Research Centre. All patients were subjected to a thorough clinical examination, pedigree analysis, and biochemical investigations [including lactate, pyruvate, creatine phosphokinase (CPK), ammonia, and other metabolic screening tests].
Neurophysiologic investigations were carried out including electroencephalogram (EEG), electromyelogram (EMG), nerve conduction velocities (NCV), complete eye evaluation including electroretinogram and visual-evoked potential, hearing test, brain MRI, and magnetic resonance spectroscopy (MRS), and pathological examination on muscle biopsy stained by the modified Gomori trichrome stain.
Genomic DNA was extracted from peripheral blood leukocytes using a standard extraction method (Miller et al., 1988). Patients were screened for seven of the most common mutations in the mtDNA (3243A>G, 3271T>C, 8344A>G, 8993T>G/C, 3256C>T, 4332G>A, and 12147G>A) using PCR-RFLP analysis. The sequence of primers and the restriction enzymes used are listed in Table 1. Seven uniplex PCR reactions were carried out in a 25 µl total volume containing 10 ng genomic DNA, 0.2 mmol/l of each dNTP (Finnzyme, European Union), 1 U Taq. Polymerase (Finnzyme, European Union), 1.2 pmol/μl of the 3243 primers, 2.4 pmol/μl of the 3271 primers, 2.4 pmol/μl of the 8344 primers, 2 pmol/μl of the 8993 primers, or 2.8 pmol/μl of the 3256, 4332, and 12147 primers. The annealing temperatures used for each amplification were 53, 52, 55, 60, 50, 56, and 55°C, respectively.
Of the 28 patients with suspected mitochondrial disorders, 17 were females and 11 were males. Patients were classified into three groups. Group 1 included four patients with suspected LS (P1–4), group 2 included two patients with suspected MELAS (P5 and 6), and group 3 included 22 patients with suspected general mitochondrial disorder (P7–28). Patients’ ages of onset ranged from 4 months to 1 year in group 1, 7 months to 9 years in group 2, and 2 months to 4 years in group 3 (mean age 1 year and 4.5 months).
P4 had 2 affected sibs who were normal until the age of 1.5 years; then, they developed fever, followed by sudden loss of acquired milestones, and died at the age of 2.5 years. P8 and P10 each had one sib with the same manifestations. P12 had two affected sibs: one with microcephaly and moderate mental retardation (IQ: 58) and the other with urethral stricture. P14 had one affected sib with mental retardation. P18 had one sib with floppiness since birth and died at 9 months of high fever; also, her mother had an abortion in the first trimester. The mother of P23 had an abortion in the second trimester. P22a and b had a family history of a matrilineal relative with epilepsy (Fig. 1a). P24 had a family history of one matrilineal relative with hydrocephalus and neural tube defect and three other relatives with mental retardation (Fig. 1b).
Patients of group 1 presented with either delayed motor and mental milestones or loss of acquired milestones, seizures, hypotonia with or without hyporeflexia, dystonia, or muscle rigidity. P3 also had eye affection manifested as ptosis, squint, and external ophthalmoplegia, whereas P4 had dysmorphic features. EEG in all patients showed either a bilateral focal epileptogenic discharge or an epileptogenic dysfunction. The results of EMG showed myopathy in two patients (P1 and P4), with axonal demyelinating neuropathy in NCV of P4. The results of brain MRI showed an abnormal signal of white matter, basal ganglia, and midbrain with central and cortical atrophic changes. All the patients had high blood lactate levels and three of them (P1, P2, and P4) showed a high lactate peak by the MRS (Table 2).
The two patients from group 2 had stroke and seizures. One patient (P5) had hypertonia and hyperreflexia, whereas the other (P6) had hypotonia and hyporeflexia. P5 also had loss of acquired milestones and hepatomegaly. The results of EEG in both patients indicated epileptogenic dysfunction. Brain MRI showed bilateral basal ganglia degeneration, white matter affection, cortical changes, and cerebellar atrophy. P5 had an abnormal visual pathway in visual-evoked potential and high blood lactate and CPK (Table 2).
Patients of group 3 (22 patients) had hypotonia with or without hyporeflexia, muscle wasting, dystonia, brisk reflexes, head nodding, drunken gait, hyperextensibility of the fingers and wrist joint, or uncontrolled urination and defecation. One patient (P11b) had hypertonia, hyperreflexia, Rocker bottom feet, and hyperextensibility of the knee joint and fingers. Other manifestations found in this group of patients were delayed mental and/or motor milestones (19/22 patients), loss of acquired milestones with or without failure of growth (3/22), mental retardation (6/22), seizures (11), eye affection (nystagmus, squint, cataract) (six), hearing loss (six), hepatomegaly with edema (one), splenomegaly (one), heart disease (three), repeated chest infection (one), dysmorphic features (three), microcephaly (one), and dysphasia (one). The results of EMG showed myopathy in 14 patients. NCV showed neuropathic changes (demyelinating polyneuropathy or peripheral polyneuropathy) (11/22). Ragged red fibers (RRF) were found in muscle biopsy stained with Gomori trichome in seven of 22 patients. EEG indicated focal epileptogenic dysfunctions in 14 of 22 patients. For all 22 patients, the results of brain MRI showed cortical changes with myelination defect, cerebral and cerebellar atrophy, basal ganglia degeneration, white matter signal with demyelination, hypogenesis of corpus callosum, thin corpus callosum, a small retrocerebellar arachnoid cyst, or demyelination around occipital horns (Fig. 2). Echo heart of two patients (P18 and P21) showed a dilated hypertrophic left ventricle and mild mitral regurge and cardiomyopathy. Patient 23 had an ECG signal of premature ventricular contraction and right axis deviation, with Echo heart indicating dilated cardiomyopathy. Biochemical investigations showed high blood lactate (16/22), high blood pyruvate (9/22), high blood CPK (10/22), high blood CPK-MB (3/22), and high serum ammonia (1/22). Six of the patients in group 3 were brothers and sisters who came from three different families (P11a and b, P19a and b, P22a and b). Each two sibs had the same manifestations, except P11a and b; one had delayed milestones and hypotonia and the other had loss of acquired milestones, failure of growth, hypertonia, and hyperreflexia (Table 2).
PCR-RFLP analyses for the detection of the seven common mitochondrial mutations were negative in the patients studied (Figs 3–9). In all cases, only a band corresponding in size to that of the fragment containing the normal nucleotide was observed: a 196 bp band for the 2343A digested fragment (Fig. 3), a 172 bp band for the 3271T fragment (Fig. 4), two bands (299 and 78 bp) for the 8344A digested fragment (Fig. 5), a 554 bp band for the 8993T fragment (Fig. 6), a 100 bp band for the 3256C fragment (Fig. 7), two bands (231 and 106 bp) for the 4332G digested fragment (Fig. 8), and a 123 bp band for the 12147G fragment (Fig. 9).
The classification of mitochondrial disease is difficult. A purely clinical classification can be helpful. However, there is often considerable clinical variability and many affected individuals do not fit neatly into one particular disease category. The patients studied were divided into three groups according to the clinical manifestations.
Four patients fulfilled the criteria of suspected LS. Their symptoms began at the ages of 3 months and 1 year, and the majority of them presented with abnormalities in the central and peripheral nervous systems without the involvement of other tissues, a feature that characterizes LS (Yang et al., 2006; Finsterer, 2008), which has a typical age of onset between 3 and 12 months (Thorburn and Rahman, 2011). The neurological features in LS included hypotonia, hyporeflexia, dystonia, muscle rigidity, and seizures (myoclonic or generalized tonic–clonic) (Tsuji et al. 2003; Thorburn and Rahman, 2011). The results of neuroimaging showed focal and necrotizing lesions of the basal ganglia or midbrain that was responsible for the neurological abnormalities, reported in other studies (Yang et al., 2006; Finsterer, 2008). One of the patients also presented with squint, ptosis, and ophthalmoplegia that were reported as brainstem, cerebral, and ophthalmologic symptoms associated with Leigh or Leigh-like syndromes (Rahman et al., 1996; Debray et al., 2007; Finsterer, 2008). Dysmorphic features were found in one patient (P4). The lactate level was increased in blood and/or cerebrospinal fluid in all four patients, a finding present in most LS cases (Finsterer, 2008).
In the two patients with suspected MELAS, the symptoms started at the age of 7 months and 9 years. The onset of MELAS was initially described to occur in the age range from younger than 2 years to older than 60 years, although almost 70% of patients presented with initial symptoms between 2 and 20 years of age (Sproule and Kaufmann, 2008). They had stroke attacks and seizures, which are invariant criteria of the disease (DiMauro and Hirano, 2010). P6 presented with stroke-like episodes of transient cortical blindness associated with dementia and hemiparesis preceded by dystonia. This is in agreement with the findings of Sproule and Kaufmann (2008). The third criterion for the diagnosis of MELAS is lactic acidosis and/or RRF (DiMauro and Hirano, 2010), and only one patient of this group (P5) had a high blood lactate level. High blood and/or cerebrospinal fluid lactic acid is an almost universal finding, occurring in 94 of 101 (94%) patients in the study of Hirano and Pavlakis (1994), seizures in 97 of 102 (96%), and stroke-like events in 106 of 107 (99%). The typical results of brain MRI of MELAS were found in terms of asymmetric lesions of the occipital and parietal lobes that mimic ischemia that are often restricted to the cortex with relative sparing of deep white matter (Sproule and Kaufmann, 2008). However, the results of the two patients indicated the presence of cortical changes in P6 that occurred in both occipital and frontal lobes, and were associated with cerebellar atrophy. In P5, the cortical atrophy was associated with bilateral basal ganglia degeneration and white matter affection.
The age of onset in group 3 ranged between 2 months and 4 years; the mean age was 1.5 years. Mitochondrial diseases may present at any age of onset (Haas et al., 2007). Seventy percent of MELAS cases have an onset at less than 2 years to more than 20 years (Sproule and Kaufmann, 2008), MERRF may occur in childhood (DiMauro and Hirano, 2009), LS typically occurs between 3 and 12 months of age and a later onset (>1 year or in adulthood) occurs in up to 25% of cases (Goldenberg et al., 2003), and NARP may occur in early childhood (Thorburn and Rahman, 2011).
The patients in group 3 did not fulfill the clinical criteria of any specific classical syndrome. Twelve patients showed multisystem involvement (brain and muscles in association with ear, eye, spleen, liver, heart, chest, or blood), reflecting, in part, the dependence on energy derived from oxidative phosphorylation in a wide variety of tissues, a feature that is a hallmark of mitochondrial diseases and may be the initial indication for the correct diagnosis (McFarland and Turnbull, 2009; Wong et al., 2010). However, they manifested with the common symptoms and signs of mitochondrial diseases that include seizures (generalized convulsion and generalized tonic–clonic), myoclonic jerks, unexplained hypotonia in newborns, infants, or young children, dystonia, basal ganglia diseases, and dilated cardiomyopathy with muscle weakness, in conjunction with other nonspecific symptoms of mitochondrial disease such as hearing loss, axonal neuropathy, and unexplained cerebral and cerebellar atrophy (Haas et al., 2007; McFarland and Turnbull, 2009). It has been reported that when the nonspecific symptoms present in combination, the likelihood of a mitochondrial disorder increases particularly if the nonspecific features involve different organ systems as in our patients (Haas et al., 2007; Debray et al., 2008).
Sixteen of the patients in group 3 had high blood lactate and/or high lactate peak on MRS and eight patients had high blood pyruvate. Despite their lack of specificity, an elevated plasma lactate or pyruvate level could be an important marker of mitochondrial disease (Haas et al., 2007).
Seven of the 22 patients, with onsets at 1 or 3 years, showed abnormal subsarcolemmal accumulations of mitochondria on skeletal muscle biopsy stained with Gomori trichrome, a unique feature of mitochondrial disease described as the ‘RRF’. However, RRFs are rarely seen in early childhood, when subsarcolemmal accumulation of mitochondria may be mild or absent. In addition, abnormal accumulation of mitochondria may be absent in patients with proven mitochondrial disease such as LS and NARP (McFarland and Turnbull, 2009; Wong et al., 2010; Thorburn and Rahman, 2011).
The nonspecific features of mitochondrial diseases makes studies of family history difficult. The clinical variability among siblings of P4, P12, P14, and P18 is a common finding in mitochondrial disease caused by mtDNA defects. This is believed to reflect the mitochondrial ‘genetic bottleneck’ (Chinnery and Schon, 2003), in which a decrease in the number of mtDNA repopulating the offspring occurs in the early stages of development and causes a sampling effect and accounts for the rapid changes in heteroplasmy levels between offspring (Turnbull et al., 2010; Yu-Wai-Man and Chinnery, 2011). Also, the variability in the symptoms between probands and their maternal relatives, similar to that found in P22a and b and P24, may be because of the presence of variable heteroplasmic levels of the mtDNA mutations between them. The presence of nonsymptomatic maternal relatives of the other patients may be because of variations in the expression of the disease or in the presence of low levels of heteroplasmic mutations (Chinnery et al., 1999; Turnbull et al., 2010).
Determining which one of dozens, if not hundreds, of genes spanning two genomes responsible for mitochondrial dysfunction in a given patient is a challenge (Haas et al., 2008). There are a number of recurrent mtDNA point mutations, including the 3243A>G transition in the mitochondrial transfer RNA Leucine1 gene (tRNALeu (UUR); OMIM#590050), which is the most common mtDNA point mutation. It accounts for more than 85% of patients with MELAS and for a large number of progressive external ophthalmoplegia patients who do not have large-scale deletions. The less common 3271T>C mutation in the same gene was found in about 10% of MELAS patients (DiMauro and Hirano, 2010; Krishnan et al., 2010). The point mutation 8344A>G in the tRNALys gene (OMIM#590060) has been found in more than 85% MERRF patients (DiMauro and Hirano, 2009). The 8993T>G/C mutations in the mitochondrial ATP synthase subunit 6 gene (MT-ATPase 6; OMIM#516060) are found in about 10–30% of individuals with maternally inherited LS (Rahman et al., 1996; Makino et al., 1998; Thorburn and Rahman, 2011), whereas the same mutations account for more than 50% in NARP patients with elevated blood lactate concentrations (Melone et al., 2004; Taylor et al., 2004; Rantamäki et al., 2005; Thorburn and Rahman, 2011).
In their last update in 4 September 2012, the MITOMAP website reported a ‘confirmed’ status for the 3256C>T and 3271T>C mutations with MELAS, the 4332G>A mutation with encephalopathy/MELAS overlapped disease, and the 12147G>A mutation with MERRF-MELAS/cerebral edema overlapped disease (http://mitomap.org/bin/view.pl/MITOMAP/ClinicalPhenotypesRNA) (MITOWEB, 2012). The confirmed status indicates that at least two or more independent laboratories have published reports on the pathogenicity of these mutations and it is generally accepted by the mitochondrial research community as being pathogenic (Bataillard et al., 2001; Sternberg et al., 2001; Melone et al., 2004; Taylor et al., 2004; Nishigaki et al., 2010). Considering the reported literature, we chose these common mutations to verify whether these mutations are also common in the Egyptian population.
The molecular analysis of the 28 patients did not indicate any of the common mutations associated with mitochondrial respiratory chain disorders. A different genetic pattern with different mutations responsible might be found in Egyptians. The mutations could be very different from that reported in the literature; thus, further sequencing of the mitochondrial DNA is recommended in order to unveil these mutations in Egyptian patients with mitochondrial disorders. Also, the low-level heteroplasmy of some mtDNA mutations in peripheral blood samples could be responsible for a false-negative result (Haas et al., 2008). The 3243A>G MELAS mutation can be found in about 50% of the cases when blood samples are used for PCR-RFLP. This is believed to be the result of the rapidly replicating ability of the blood cells, which will gradually eliminate the leukocytes with a higher mutation load (Sue et al., 1998). However, patients with the 8344A>G MERRF mutation have a high percentage of mutant mtDNA (90%) in blood and muscle and the degree of heteroplasmy is evenly distributed between different organs (Lertrit et al., 1992; Tanno et al., 1993; Oldfors et al., 1995; Brinckmann et al., 2010). Furthermore, white blood cells or any other tissue type can be used to test for the 8993T>G/C mutations (Thorburn and Rahman, 2011) as they do not show any significant variation in the mutation load among tissues (White et al., 1999).
Establishing a specific diagnosis in a patient with the suspected mitochondrial disease is a complex endeavor that requires the integration of clinical assessments, family history, electrophysiological investigations, biochemical testing, histopathological examination, and molecular testing. Close collaboration between primary clinicians, geneticists, pathologists, other clinical specialists, and diagnostic laboratories with expertise in mitochondrial biochemical and molecular testing is critical to maximize the likelihood of establishing a correct diagnosis. The negative molecular results of the seven chosen common mutations in our study could be a result of the presence of other mutations in either of the two genomes or a result of the low heteroplasmic levels of the mtDNA mutations in leukocytes.
Conflicts of interest
There are no conflicts of interest.
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Keywords:© 2013 Middle East Journal of Medical Genetics
common mtDNA point mutations; mitochondrial respiratory chain disorders; molecular diagnosis