since the first mutations in mtdna were described , more than 200 pathogenic point mutations and rearrangements have been found in patients with mitochondrial cytopathies .
mitochondrial trna genes are hotspots for mutations associated with multi - organ involvement , making them a major contribution to mitochondrial disorders caused by mtdna mutations , due to their essential role in mitochondrial protein synthesis .
we report the second case of a patient carrying a de novo m.5540g > a mutation affecting the mitochondrial trna gene .
this patient presented with an early onset and severe progressive encephalopathy associated with sensorineural hearing loss .
we present the case of a 10-year - old female , born after an uneventful pregnancy as the second child of a non - consanguineous healthy couple .
her family history did not reveal other family members afflicted with neuromuscular or hearing disorders .
she had normal neurodevelopment until age 6  years . at that time , she presented with atonic  myoclonic epilepsy , associated with ptosis of the right eye . brain mri revealed bilateral basal ganglia calcifications .
initially , the epilepsy was treated successfully with zonisamide . at age 7  years , the patient exhibited progressive gradual decline of truncal and appendicular ataxia , tremor , spasms , muscular weakness , regression of gross and fine motor skills , and bilateral sensorineural hearing loss .
carnitine , coenzyme q10 ( coq10 ) and a b vitamin complex supplementation were trialed .
however , no improvement in symptomatology was observed prompting their discontinuation . at age 10  years
clinical examination was remarkable for low weight ( 17.1  kg , <  3rd percentile ) , short stature ( 125.7  cm , 2nd percentile ) , axial hypotonia , appendicular hypertonia , contractures of the fingers , muscle weakness , hyperreflexia of the lower extremities , decreased muscle mass , ataxic gait , dysmetria , horizontal nystagmus , and bilateral ptosis greater on the right upper lid .
ophthalmologic examination found a symmetric diffuse granular retinal pigment epitheliopathy from the macular areas to the far periphery , without pigment migration into the retina .
initial laboratory studies revealed normal urine organic acid analysis , plasma amino acids , and acylcarnitine profile .
spectrophotometric analysis of mitochondrial respiratory chain enzymatic activities on skeletal muscle , , ,   showed partial reductions in complex ii+iii and rotenone sensitive complex i+iii activities ( table  1 ) .
analysis of a panel of common mtdna point mutations and single deletions performed in blood did not detect deleterious mutations .
comprehensive analysis of the circular mitochondrial genome by ngs revealed a 25.6% heteroplasmic mutation , m.5540g > a , in blood .
the m.5540g > a is located in the anti - codon stem of mitochondrial trna and changes the original g : c pairing to a : c mispairing ( fig .
analysis of the mitochondrial genome from the blood of the patient 's mother did not detect the mutation .
mutations in the mt - tw gene coding for the mitochondrial trna are associated with diverse phenotypes , such as neonatal onset mitochondrial encephalomyopathy , mitochondrial myopathy , leigh syndrome , dementia and chorea , and a neurogastrointestinal syndrome . here
we present the case of a female patient harboring the m.5540g > a transition involving the mt - tw gene .
however , the onset of neurological disease occurred in the second decade of life . that subject exhibited sca and sensorineural hearing loss , peripheral neuropathy , dysmetria , dysdiadochokinesia , elicited nystagmus , areflexia of lower extremities , pes cavus , muscle hypotrophy , apallesthesia , and lactic acidosis .
brain mri showed marked atrophy of brain and cerebellum , diffuse white matter abnormalities and basal ganglia hypointensities .
that patient was reported to have a 98% heteroplasmy level on skeletal muscle , and the authors demonstrated by single fiber pcr that the cox - negative fibers had a significantly higher percentage of heteroplasmy than their surrounding normal fibers .
a comparison of clinical features between these two patients reveals some remarkable differences ( table  2 ) . despite having a lower heteroplasmy in skeletal muscle ( 51.4% ) ,
our patient developed signs of neurodegeneration and cerebellar dysfunction at an earlier age , and exhibited a worse clinical course and a faster clinical progression .
this finding suggests that the level of heteroplasmy in skeletal muscle may not always be a good predictor of the severity of the neurological disease .
in addition , our patient may have a higher level of heteroplasmy in the central nervous system .
likewise , the patient from the previous report may have had a different tissue distribution of the mutation heteroplasmy , leading to later onset and milder clinical phenotype .
furthermore , other factors such as nuclear and environmental modifiers and specific mitochondrial haplotypes may be responsible for the observed clinical heterogeneity .
it is noticeable that in our case muscle morphology was more sensitive than the analysis of mitochondrial respiratory chain enzymatic activities .
although we have been able to demonstrate a deficiency in complex i  +  iii activity by running a complete panel of mitochondrial enzymes , the reduction did not meet a major criterion ( residual activity of <  20% ) based on walker modified criteria .
this finding may be due to the fact that , with a relatively low mutational load in skeletal muscle , a severe biochemical defect may be present only in a few muscle fibers and may not be detectable by a spectrophotometric assay .
moreover , our patient has certain findings not described in silvestri 's paper , such as epilepsy , hypertonia , muscle spasms , ptosis , and pigmentary retinopathy .
the earlier report emphasized dementia late in the third decade of her life and severe cortical and cerebellar atrophy .
the brain mri in our proband did not reveal these findings , perhaps reflecting that she is younger , or that these features may require time to evolve .
both patients shared several neurologic features including childhood - onset sensorineural hearing loss and multiple signs of cerebellar dysfunction ( ataxia , nystagmus , tremor , dysmetria , and dysdiadochokinesia ) .
cox deficiency and cox negative fibers are the most frequent biochemical and histochemical features of mt - tw mutations , including silvestri 's patient , , , , , , , , , .
although our proband did not have cox deficiency on mitochondrial enzyme assays , she had reduced staining for cox in type 2 fibers .
these findings may reflect the fact that two cox subunits , coi and coiii , have a relatively higher content of tryptophan ( 3.1% and 4.6% , respectively ) than the average content of the other mtdna - encoded subunits ( 2.6% ) .
the absence of ragged red fibers in our patient could be explained by a muscle biopsy done at a young age .
multiple sequence alignment with clustalw reveals that this mutation affects a highly conserved nucleotide in the anticodon arm stem ( fig .
1 ) . according to rnaalifold analysis   ( http://rna.tbi.univie.ac.at/cgi-bin/rnaalifold.cgi ) , this nucleotide participates in a highly conserved base pair .
the rnafold software   ( http://rna.tbi.univie.ac.at/cgi-bin/rnafold.cgi ) , predicted that this transition may alter the secondary structure of the trna .
currently , this mutation is not registered in mtdb   ( http://www.mtdb.igp.uu.se/ ) and is reported as pathogenic in mitomap ( http://www.mitomap.org/mitomap ) .
it is classified as definitively pathogenic with a score >  30 according to the scoring criteria devised by scaglia and wong . in conclusion
, this case further supports the pathogenic effect of the m.5540g > a mutation , and its association with a neurological phenotype .
moreover , the shared clinical presentation of cerebellar ataxia and sensorineural hearing loss could suggest a discernible phenotype that should be considered during the diagnostic assessment of patients with genetic ataxias .
tissue distribution of mutation heteroplasmy , and other genetic modifiers may be responsible for the clinical heterogeneity observed in these two reported patients .
the earlier age of onset and more severe clinical course in our proband expands the neurological phenotype associated with this mtdna mutation .
 we report a patient harboring a de novo m.5540g > a mutation affecting the mt - tw gene coding for the mitochondrial tryptophan - transfer rna . 
 this patient presented with atonic  
 myoclonic epilepsy , bilateral sensorineural hearing loss , ataxia , motor regression , ptosis , and pigmentary retinopathy . 
 our proband had an earlier onset and more severe phenotype than the first reported patient harboring the same mutation . 
 we discuss her clinical presentation and compare it with the only previously published case . 
Introduction
Case report
Results
Discussion
Conflict of Interest