respiratory chain defects ( rcd ) are usually phenotypically related to heterogeneous clinical features , ranging from fatal infantile multisystem syndromes to encephalomyopathies or isolated myopathies sometimes associated with cardiomyopathies .
hypotonia , lactic acidosis , cardiorespiratory failure and severe psychomotor delay are the most frequently reported features in paediatric patients , while myopathy , associated to central nervous system involvement ( hearing loss , pigmentary retinopathy , seizures , ataxia , polyneuropathy , rarely movement disorders ) is a main characteristic of the adult - onset pathologies .
respiratory chain defects are either related to mitochondrial dna mutations , or to abnormalities in nuclear genes linked to mitochondrial function .
complex i ( ci , nadh dehydrogenase ubiquinone  ubiquinol reductase ) is the largest complex of the respiratory chain .
it catalyses the transfer of electrons from nadh to coenzyme q10 , and consists of 45 subunits , seven of which ( nd1nd6 , nd4l ) encoded by the mitochondrial genome . in mitochondrial disorders ,
isolated ci deficiency is relatively frequent   , usually associated with severe , early - onset , multisystem phenotype . due to the enzyme complexity , in almost half of the cases of ci defect ,
only few mutations in the mitochondrial dna ( mtdna)-encoded nd2 subunit ( ec:1.6.5.3 ) have been reported , usually associated with leigh syndrome , and leber 's hereditary optic neuropathy ; a single patient has been reported carrying a 2-bp deletion in mtnd2 gene and suffering from severe exercise intolerance . here
we report a new mutation in the mtnd2 gene in a patient with a severe and isolated ci defect showing a relatively mild phenotype characterized by exercise intolerance and lactic acidosis .
the patient is a 21-year - old man , the first born after uncomplicated pregnancy and delivery from healthy unrelated parents .
first symptoms became evident at the age of 7  years , when he began to complain of overall fatigability presenting exclusively during physical activities , and worsened by exposure to cold temperatures . in the late five years , the exercise intolerance became so severe he could not keep up with his schoolmates when playing and running , requiring 30 to 60  min to regain the overall strength .
the progressive fatigability eventually prevented him from riding a bike , then he became unable to carry ordinary burdens ( i.e. backpack , books ) while the tolerable walking distance gradually shortened to 150  m. neither cognitive nor behavioural changes were described ; he did not report any hearing , vision , speech impairment , he never lost consciousness , and he never experienced any selective muscle group weaknesses nor myoglobinuria . starting from the age of 17  years , he began to exhibit frequent vomiting while exercising , symptoms that urged the patient 's family to search for medical help .
the first clinical evaluation performed at 20  years of age showed asthenic habitus , no dysmorphic facial features but he showed high - arched palate and malocclusion of teeth , normal head circumference , low body mass index ( bmi , 18 
clinical tests for myasthenia , prostigmine test , anti - achr and anti - musk antibodies were negative .
however , he referred general weakness during arm uplifting >  60  s. and five squats .
the psychological testing and psychiatric status were normal ( full - scale iq  =  97 , wais - iv ) .
electrophysiological assessment ( electroencephalogram , multimodal evoked potentials , eng , emg with repetitive stimulation ) and brain mri with spectroscopy were normal .
thyroid status and routine blood tests for renal and liver function were within normal ranges , so as ck values ( 190  u / l ) .
lactate at rest were increased in the blood ( 9.9  mmol / l , n.v .
<  2  mmol / l ) increasing at 14,4  mmol / l after exercise , while normal in cerebrospinal fluid .
electrocardiogram and echocardiogram were normal , while 24  h - holter ecg detected a supraventricular tachycardia ( 190/min ) during minor physical activity ( e.g. slow walking ) , with normal heart rate at rest ( 80/min ) and ergometry test .
respiratory assessment ( clinical examination , spirometry , arterial blood gas analysis ) was normal .
since a mitochondrial disorder was suspected , the patient underwent a muscle biopsy and was afterwards put on oral antioxidant therapy ( coenzyme q10 180  mg per day and vitamin b complex ) .
after the starting of antioxidant therapy , he showed an improvement of his condition , with a better tolerance of physical activity better and a decrease of his overall fatigability with shortening of the post - exercise recovery time ; no effects were reported on exercise tolerance .
histological and ultrastructural assessment of the muscle biopsy were performed using standard histological , histochemical and electron microscopy techniques .
mitochondrial respiratory chain ( mrc ) enzymes activity was assayed by standard spectrophotometric techniques in muscle homogenate and in digitonin - treated cultured fibroblasts obtained from skin biopsy .
each mrc enzyme specific activity was normalized to that of citrate synthase ( cs ) , a standard marker of cellular mitochondrial content .
normal activity range is expressed as mean value  standard deviation ; residual activity was expressed in percentage .
the entire mtdna was pcr - amplified and sequenced as described in bugiani et al . .
restriction fragment length polymorphism ( rflp ) analysis was used to confirm and quantify the mutation using the nlaiv enzyme ( neb ) .
quadriceps muscle biopsy disclosed the presence of numerous ragged red - cox - positive fibres , with accumulations enlarged mitochondria and mitochondrial proliferations ( fig .
the electron microscopy confirmed the presence of numerous and enlarged mitochondria , often with abnormal proliferating cristae and rounded osmiophilic inclusions ( fig .
biochemical assays of mrc complexes performed on muscle homogenate showed a severe isolated ci deficiency equal to 10% of control mean with very high cs activity equal to 345% of control mean ; all mrc activities was normal on patient 's fibroblasts ( table 1 ) .
we performed the sequence analysis of entire mtdna , classified into haplogroup j1c3 by haplogrep2 inherited from the mother , as confirmed by the same analysis performed on her dna , and identified a new mutation in mtnd2 gene ( m.4831 g  >  a ) that causes the substitution of the glycine in position 121 with an aspartic acid ( p.gly121asp ) in the protein , in a site between two intermembrane domains .
the mutation has never been reported , the p.gly121asp change scored very highly for likelihood to be deleterious according to ad - hoc softwares for pathogenicity prediction ( damaging for polyphen2 : p  =  1.000 ; panther : 0.96 ; mutpred : 0.969 ; sift and mutationtaster ) and the amino - acid involved is highly conserved in the phylogenies ( fig .
pcr - rflp analysis performed to quantify the mutation in different tissues using the nlaiv enzyme ( fig .
2b ) showed the presence of the m.4831 g  >  a change in the 95% of mitochondrial genomes from patient 's muscle , in the 40% of genomes from urinary tract cells and only in <  5% of genomes from patient 's peripheral blood lymphocytes .
the mutation was absent in fibroblasts obtained from skin biopsy as in the tissues ( blood and urine ) from the healthy mother and younger brother ( fig .
isolated ci deficiency is a frequent cause of mitochondrial dysfunction , usually related to severe and early - onset multisystem phenotype and associated with mutations in nuclear genes coding for ci structural proteins .
we report a novel mutation in mitochondrial mtnd2 gene coding for a ci subunit , characterized by a mild phenotype with exclusive muscular involvement presented as exercise intolerance and high blood lactate at rest .
vomiting while exercising might be due to frequent physiological causes or rare somatic causes , and it is often a hallmark of exercise intolerance . in our patient coenzyme q10 therapy could ameliorate the capability to endure physical activity , but was unable to treat the overall fatigability .
the severe ci deficiency observed in patient 's muscle homogenate is related to the almost homoplasmic mutation in this tissue , while in fibroblasts , where the mutation was absent , the activity was normal .
this evidence confirms the pathogenicity of the mutation which , by changing an amino - acid highly preserved between species that links two intermembrane domains , probably affects their stability and function .
furthermore , the high mutation load on muscle tissue can explain the onset in childhood and the relatively benign course of the disease involving only skeletal muscle , but raises the question of how and when the specific tissue segregation ensued in this patient .
the absence of the mutation in the healthy mother and brother suggests the exclusive presence of a lower mutation load in the ovarian cells or a de novo genesis in our patient .
extreme exercise intolerance and isolated mitochondrial myopathy has been reported in mitochondrial dna mutations in at least 5 genes : mtcytb mtnd2 , mtnd4 , mtnd5 , and trnas .
our case resembles that of several mutations in mtcytb , another mtdna gene that encodes for the cytochrome b , a subunit of complex iii , which are often sporadic , in which a pure myopathy with exercise intolerance has been described , , . since now only few patients showing an exclusively mild muscular phenotype have been described carrying mutations in mtnd4 and mtnd5 genes coding for ci ,   and only one patient has been reported with a phenotype similar to our patient , characterized by severe exercise intolerance and lactic acidosis , due to a deletion of 2  bp in mtnd2 gene . in all this patients
the muscle biopsy showed ragged - red and cox - positive fibres ; the mutations were present at high level only in muscle tissue , as seen in our patient .
the accumulation of mitochondria is present in a large number of muscle fibres in particular in type 2 fibres , which show a peculiar large accumulation of mitochondria positive for cox and sdh staining that correlate with the biochemical data of a significative increment of citrate synthetase activity and a normal cox activity .
nevertheless the presence of mitochondrial proliferation with enlarged organelles containing osmiophilic inclusions and abnormal cristae confirm the severe involvement of mitochondria in muscle in conclusion , in the presence of isolated exercise intolerance with high blood lactate , histopathological signs of mitochondrial myopathy and defects of ci activity , the sequence analysis of mitochondrial dna should be performed .
 to date , only few mutations in the mitochondrial dna ( mtdna)-encoded nd2 subunit of complex i have been reported , usually presenting a severe phenotype characterized by early onset encephalomyopathy and early death . in this report 
 , we describe a new mutation in the mtnd2 gene in a 21-year - old man with a mild myopathic phenotype characterized by exercise intolerance and increased plasma lactate at rest . 
 electromyography and brain nmr were normal , and no cardiac involvement was present . 
 muscle biopsy showed a massive presence of ragged red  cox - positive fibres , with enlarged mitochondria containing osmiophilic inclusions . 
 biochemical assays revealed a severe isolated complex i deficiency . 
 we identified a novel , heteroplasmic mutation m.4831 g  >  a in the mtnd2 gene , causing the p.gly121asp substitution in the nd2 protein . 
 the mutation was present in the 95% of mitochondrial genomes from patient 's muscle tissue , at a lower level in cells from the urinary tract and at a lowest level in lymphocytes from patient 's blood ; the base substitution was absent in fibroblasts and in the tissues from proband 's healthy mother and brother . 
 the specific skeletal muscle tissue involvement can explain the childhood - onset and the relatively benign , exclusively myopathic course of the disease . 
Introduction
Case report
Materials and methods
Results
Discussion